Modelling aspects of neurodegeneration in Saccharomyces cerevisiae
A thesis presented for the degree of Doctor of Philosophy by
Mathew Danger Traini
School of Biotechnology and Biomolecular Sciences University of New South Wales 2009
i Contents
ABSTRACT...... V ACKNOWLEDGEMENTS...... VII ABBREVIATIONS...... VIII 1: INTRODUCTION...... 1
1.1: NEURODEGENERATION AND PROTEIN AGGREGATION ...... 1 1.2: ALZHEIMER'S DISEASE (AD) ...... 2 1.2.2: Tangles are composed of hyperphosphorylated tau ...... 2 1.2.3: Plaques are composed of amyloid beta ...... 3 1.2.4: Proteolytic processing of APP and production of amyloid beta...... 4 1.2.5: The amyloid cascade hypothesis of Alzheimer's Disease...... 6 1.2.6: Refining the amyloid hypothesis: soluble A oligomers versus plaques ...... 8 1.2.7: Intracellular and extracellular pools of A ...... 10 1.3: PARKINSON'S DISEASE ...... 11 1.3.1: Clinical symptoms of Parkinson's Disease...... 11 1.3.2: alpha-synuclein is the major component of Lewy Bodies...... 12 1.3.3: Environmental factors and Parkinson's Disease ...... 13 1.3.4: Pesticides and Parkinson’s Disease ...... 16 1.3.5: Paraquat...... 16 1.3.6: Rotenone...... 17 1.3.7: Organochlorines ...... 18 1.3.8: Multiple hit models of Parkinson's Disease ...... 21 1.4: SACCHAROMYCES AS A MODEL ORGANISM ...... 22 1.4.1: Yeast models of neurodegeneration ...... 24 1.4.2: Yeast models of Parkinson's Disease: -synuclein...... 25 1.4.3: Yeast models of Alzheimer's Disease: APP, tau and A ...... 26 1.5: AIMS OF THIS STUDY...... 29 CHAPTER 2: MATERIALS AND METHODS...... 31
2.1: GENERAL MATERIALS ...... 31 2.2: ESCHERICHIA COLI MEDIA, STRAINS, SELECTION AND STORAGE ...... 31 2.3: S. CEREVISIAE MEDIA AND STORAGE...... 32 2.4: S. CEREVISIAE STRAINS ...... 33 2.5: SYNTHETIC OLIGONUCLEOTIDES...... 33 2.6: GENERAL LAB PROCEDURES ...... 34 2.7: GENERAL MOLECULAR BIOLOGY PROCEDURES ...... 34 2.7.1: Small scale plasmid DNA miniprep purification...... 34 2.7.2: Large scale plasmid DNA preparation...... 34 2.7.3: Agarose gel electrophoresis...... 35 2.7.4: Determination of DNA concentration...... 35 2.7.5: Polymerase Chain Reaction (PCR)...... 35 2.7.6: Restriction digestion ...... 36 2.7.8: Ligation of DNA ...... 36 2.7.9: Creation of chemically competent E. coli ...... 36 2.7.10: Transformation of E. coli ...... 37 2.8: CONSTRUCTION OF PLASMIDS ...... 37 2.8.1: Construction of pUG34-GFP and pUG36-GFP ...... 39 2.8.2: Construction of pUG23GAL1 and pUG35GAL1...... 39 2.8.3: Construction of pUG23GAL1/A 1-42 and pUG35GAL1/A 1-42...... 39 2.8.4: Construction of pUG35GAL1/A 1-42-EP ...... 39 2.8.5: Construction of pUG35GAL1/A 1-40 ...... 40 ii 2.8.6: Construction of pUG34-GFP/A 1-42...... 41 2.8.7: Construction of pUG35GAL1/wt-tau and pUG35GAL1/P301L-tau...... 42 2.9: TRANSFORMATION OF S. CEREVISIAE ...... 42 2.9.1: Transformation of individual S. cerevisiae strains...... 42 2.9.2: Transformation of 96-well plate arrays of S. cerevisiae strains...... 42 2.10: SCREENING OF 96-WELL PLATE ARRAYS FOR A -GFP FLUORESCENCE AND LOCALISATION ...... 43 2.11: BIOINFORMATIC ANALYSIS OF S. CEREVISIAE MUTANT GENES ...... 44 2.12: PROTEIN EXTRACTION FROM S.CEREVISIAE ...... 44 2.13: DETERMINATION OF PROTEIN CONCENTRATION ...... 45 2.14: POLYACRYLAMIDE GEL ELECTROPHORESIS ...... 45 2.15: ELECTROBLOTTING OF SDS-PAGE GELS...... 45 2.16: WESTERN BLOTTING AND ENHANCED CHEMILUMINESCENCE (ECL) DETECTION ...... 45 2.17: FLUORESCENCE MICROSCOPY AND STAINING ...... 46 3: A FLUORESCENCE-BASED REPORTER OF INTRACELLULAR AMYLOID-BETA AGGREGATION IN SACCHAROMYCES CEREVISIAE ...... 48
3.1: INTRODUCTION AND AIM...... 48 3.2: RESULTS ...... 49 3.2.1: An A 1-42-GFP fusion protein is not fluorescent in yeast...... 49 3.2.2: A less amyloidogenic mutant form of A 1-42-GFP is fluorescent...... 52 3.2.3: An A 1-40-GFP fusion protein exhibits fluorescence...... 55 3.2.4: Fluorescence levels of GFP, A 1-40-GFP and A 1-42-GFP are distinguishable via flow cytometry...... 56 3.2.5: Lack of A 1-42-GFP fluorescence is not due to lack of translation, cleavage, or destruction of the fusion protein ...... 58 3.2.6: Expression of A 1-40-GFP or A 1-42-GFP is not detrimental to the growth of yeast ...62 3.2.7: Expression of unfused A 1-42 does not inhibit growth, and the peptide can not be detected...... 63 3.2.8: Exposure to guanidine hydrochloride results in appearance of A 1-42-GFP fluorescence ...... 64 3.2.9: The anti-amyloidogenic effect of guanidine hydrochloride acts through a Hsp104p- independent mechanism ...... 65 3.3: DISCUSSION ...... 67 3.3.1: Comparison with other Saccharomyces cerevisiae-based models of A aggregation...68 3.3.1.1: An A -GFP fusion S. cerevisiae model ...... 68 3.3.1.2: A -Sup35p fusion S. cerevisiae models...... 70 3.3.1.3: Aggregation of A 1-42/Sup35p compared to A 1-42-GFP ...... 71 3.3.1.4: Aggregation of A 1-40/Sup35p and A 1-40-GFP fusions...... 72 3.3.1.5: Guanidine hydrochloride, Hsp104 and A 1-42...... 72 3.3.1.6: Toxicity of A 1-42/Sup35p and A 1-42-GFP fusions...... 74 3.3.1.7: Comparative strengths and weaknesses of A 1-42/Sup35 and A 1-42-GFP fusion systems ...... 75 4: A GENOME-WIDE SCREEN FOR MODIFIERS OF A 1-42 AGGREGATION AND LOCALISATION ...... 76
4.1: INTRODUCTION AND AIMS...... 76 4.2: RESULTS ...... 78 4.2.1: Transformation of the Saccharomyces genome knockout collection ...... 78 4.2.2: Screening for A 1-42-GFP fluorescence and localisation...... 78 4.2.3: Tricarboxylic acid cycle and oxidative phosphorylation...... 82 4.2.4: Chromatin remodelling and transcriptional regulation ...... 84 4.2.5: Regulation of phospholipid metabolism: ino2 , ino4 and scs2 ...... 85 4.2.6: Phosphatidylcholine synthesis: cho2 and opi3 ...... 88 4.2.7: icy2 ...... 100
iii 4.2.8: slg1 and osmolarity sensing...... 102 4.2.9: Dubious ORFs...... 103 4.3: DISCUSSION ...... 105 4.3.1: Comparison to genome-wide screens in S. cerevisiae ...... 106 4.3.2: Screens in Drosophila: involvement of chromatin remodelling and transcriptional regulation...... 107 4.3.3: Mitochondrial dysfunction and A 1-42-GFP fluorescence ...... 110 4.3.4: Phospholipid metabolism and lipid droplets ...... 111 4.3.5: A single protease results in A 1-42-GFP fluorescence...... 116 4.3.6: Future directions...... 116 5: EFFECTS OF ENVIRONMENTAL RISK FACTORS ASSOCIATED WITH PARKINSON’S DISEASE ON A SACCHAROMYCES MODEL OF -SYNUCLEIN PATHOBIOLOGY ...... 121
5.1: INTRODUCTION AND AIMS...... 121 5.2: RESULTS ...... 122 5.2.1: ROS generating chemicals enhance -synuclein toxicity ...... 122 5.2.2: Expression of -synuclein leads to an increase in endogenous production of ROS....123 5.2.3: Treatment with the antioxidant compounds ascorbate and -tocopherol does not reduce -synuclein toxicity...... 125 5.2.4: The pesticide rotenone does not enhance -synuclein toxicity...... 127 5.2.5: The pesticide dieldrin enhances -synuclein toxicity ...... 129 5.2.6: Aluminium enhances -synuclein toxicity ...... 130 5.2.7: Mutants of the phosphatidylcholine biosynthesis pathway display altered -synuclein- GFP localisation ...... 132 5.3: DISCUSSION ...... 139 5.3.2: Enhancement of -synuclein toxicity by aluminium ...... 139 5.3.3: Environmental factors: enhancement of -synuclein toxicity via metals and pesticides ...... 142 5.3.4: Phospholipid synthesis, lipid droplets and -synuclein ...... 144 6: APPENDIX...... 148 REFERENCES...... 156
iv Abstract
The neurodegenerative disorders Alzheimer’s Disease (AD) and Parkinson’s Disease (PD) are characterised by the accumulation of misfolded amyloid beta 1-42 peptide (A 1-42) or -synuclein, respectively. In both cases, there is extensive evidence to support a central role for these aggregation-prone molecules in the progression of disease pathology. However, the precise mechanisms through which A 1-42 and -synuclein contribute to neurodegeneration remain unclear. Organismal, cellular and in vitro models are under development to allow elucidation of these mechanisms.
A cellular system for the study of intracellular A 1-42 misfolding and localisation was developed, based on expression of an A 1-42-GFP fusion protein in the model eukaryote Saccharomyces cerevisiae. This system relies on the known inverse relationship between GFP fluorescence, and the propensity to misfold of an N-terminal fusion domain. To discover cellular processes that may affect the misfolding and localisation of intracellular A 1-42, the A 1-42-GFP reporter was transformed into the S. cerevisiae genome deletion mutant collection and screened for fluorescence. 94 deletion mutants exhibited increased A 1-42-GFP fluorescence, indicative of altered A 1-42 misfolding. These mutants were involved in a number of cellular processes with suspected relationships to AD, including the tricarboxylic acid cycle, chromatin remodelling and phospholipid metabolism. Detailed examination of mutants involved in phosphatidylcholine synthesis revealed the potential for phospholipid composition to influence the intracellular aggregation and localisation of A 1-42.
In addition, an existing S. cerevisiae model of -synuclein pathobiology was extended to study the effects of compounds that have been hypothesized to be environmental risk factors leading to increased risk of developing PD. Exposure of cells to aluminium, dieldrin and compounds generating reactive oxygen species enhanced the toxicity of - synuclein expression, supporting suggested roles for these agents in the onset and development of PD. Expression of -synuclein-GFP in phosphatidylcholine synthesis
v mutants identified in the A 1-42-GFP fluorescence screen resulted in dramatic alteration of -synuclein localisation, indicating a common involvement of phospholipid metabolism and composition in modulating the behaviours of these two aggregation-prone proteins.
vi Acknowledgements
Firstly I must thank my supervisor Ian Dawes for allowing me to work in his lab on what must have seemed like some slightly insane ideas, and for encouraging those ideas through to fruition (even if the fruit was occasionally rotten). Also many thanks for providing financial support where the university could not, or would not.
Enormous thanks also go to my co-supervisor Gabriel Perrone. His encyclopaedic knowledge of Saccharomyces, penchant for all-encompassing A0-sized biochemical models and limitless supply of dirty jokes have all been essential supervisorial qualities. There was never a time when I felt that I couldn’t walk into your office and discuss any part of my research with you…except immediately after the consumption of a tuna sandwich. I think it’s time to open that bottle of pineapple juice now.
Thank you to the laboratories of Susan Lindquist, Ian Macreadie, Ben Herbert, Joel Goodman and Jurgen Gotz for providing technical assistance, reagents and useful discussions.
Thank you to the members of the “Dawes Lab Massive” for all the advice, troubleshooting, coffee, larfs and centrifuge tube cocktails, especially Chong Han, Shixiong, Duncan, and Nadia. Also a special thanks to Suresh Nair for all the hard work and permanent eye damage incurred during the knockout screen.
Thank you to my parents for not only encouraging my misguided interests in science from as early an age as I can remember, but also for enduring thousands of hours of ranting about anything and everything related to this project. I promise I’ll stop now.
Finally, I reserve my deepest gratitude for my wife Christina. Without your limitless encouragement, patience, good humour and love, this would have all been utterly impossible. This work is as much yours as it is mine, and it is to you that I dedicate this thesis. Looking forward to our “post-PhD” life together!
vii Abbreviations
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viii &. &7 ( &# &% &+ &' ( )+ ( 8$ ( ( 9 :
Designations of Saccharomyces cerevisiae genes follow standard naming conventions. These may be summarised as:
ABC1 The dominant allele for the ABC1 gene abc1 The recessive allele for the ABC1 gene abc1 Deletion of all or part of the ABC1 gene Abc1p The ABC1 protein
ix 1: Introduction
1.1: Neurodegeneration and protein aggregation The correct folding of proteins is essential to avoid the loss of wild-type function and the gain of unintended, potentially toxic functions. Cells have evolved complex mechanisms to allow the efficient folding of nascent proteins, and where this is not possible, to enable the rapid recognition, refolding, or degradation of misfolded proteins.
The cells of the brain appear to be particularly sensitive to the deleterious effects of misfolded proteins. An emerging understanding of the molecular basis of a diverse range of neurodegenerative diseases has identified misfolded proteins as being central to their pathologies (reviewed in Soto and Estrada, 2008; Morimoto, 2008).
Table 1.1 lists some neurodegenerative diseases associated with protein aggregation. Alzheimer's Disease and Parkinson's Disease represent the most commonly occurring forms of dementia and movement disorders, respectively, and it is these disorders which are addressed below.
Table 1.1: Summary of key clinical, epidemiological and molecular features of neurodegenerative diseases linked to misfolded proteins. Adapted from (Soto and Estrada, 2008).
Disease Clinical features Misfolded/aggregated protein(s) - ; 0 %- # # %- & ( , - ) ) (
1 1.2: Alzheimer's Disease (AD) Alzheimer's Disease (AD), named after the psychiatrist Alois Alzheimer who first described its symptoms in 1907, is a progressive, irreversible neurodegenerative disorder. It is the most commonly occurring neurodegenerative disorder, affecting approximately 2.32 million US citizens, with the risk of developing AD doubling every five years beyond 65 years of age (Brookmeyer, et al., 1998). The most prominent symptoms of the disease are memory loss (particularly of more recent events) and cognitive impairment, which affects reasoning skills, judgement, personality and language. The appearance and progression of these symptoms is debilitating, inevitably leading to the patient becoming totally dependent on a carer's assistance. Histopathologically, AD is characterized by the appearance of two types of proteinaceous lesions, which are the hallmarks of the disease: neurofibrillary tangles and senile plaques (reviewed in LaFerla and Oddo, 2005).
1.2.2: Tangles are composed of hyperphosphorylated tau One of the two distinctive brain lesions originally used by Alois Alzheimer to classify AD are the neurofibrillary tangles (NFTs) (Figure 1.1a). NFTs may be observed in the brains of aged healthy individuals, but their prevalence is greatly increased in AD patients. NFTs consist of a mixture of paired helical filaments (PHF) and straight filaments, and may occur throughout affected neurons including the dendrites, axons and synaptic terminals. The major component of the helical filaments is the protein tau, encoded by the MAPT (microtubule-associated protein) gene. In healthy tissue, tau remains soluble and is involved in the stabilization of microtubules and in regulating their assembly. In AD, hyperphosphorylation of tau by a range of kinases causes tau to lose its affinity for microtubules, and promotes formation of insoluble PHFs and insoluble, fibrous neurofibrillary tangles (Grundke-Iqbal, et al., 1986; Goedert, et al., 1992, and reviewed in Wang and Liu, 2008).
Abnormal aggregation of tau into filaments also occurs in a number of other neurodegenerative disorders, including frontotemporal dementia, Pick disease and progressive supranuclear palsy and its role in the progression of AD has been extensively studied (reviewed in Iqbal and Grundke-Iqbal, 2008). While not discounting the
2 importance of tau in the pathology of AD, this introduction will focus on the role of amyloid plaques and amyloid beta in this disease (see below).
a
b
Figure 1.1: Senile plaques and tangles from the brain of an AD patient. Amyloid plaques (a) and neurofibrillary tangles (b) visualised immunohistochemically via staining with anti-A 1-42 or anti-PHF1 antibodies, respectively. Scale bar represents 62.5 μm. Figure adapted from LaFerla and Oddo, 2005).
1.2.3: Plaques are composed of amyloid beta Along with neurofibrillary tangles, the appearance of dense extraneuronal senile plaques is the other major histopathological hallmark of AD (see Figure 1.1b). A major step towards understanding the molecular pathology of AD came with the biochemical characterization of these extraneuronal plaques, and the elucidation of the amino acid sequence of their major proteinaceous component, the amyloid- peptide (Glenner and Wong, 1984). 3 Amyloid- (A ) is a hydrophobic polypeptide, with a length ranging between 39 to 42 amino acids. The newly discovered sequence of A , and the realization that this was the same polypeptide present in the vascular amyloid deposits of Downs Syndrome brains (Glenner and Wong, 1984), rapidly led to the identification of the amyloid precursor protein (APP) gene and its mapping to chromosome 21 (Goldgaber, et al., 1987; Tanzi, et al., 1987; Kang, et al., 1987).
APP is a large 753 amino acid transmembrane protein, trafficked through the secretory pathway and localized predominantly to the plasma membrane. APP has also been found in the endosomal compartments, ER and Golgi, depending on cell type (Benowitz, et al., 1989; McGeer, et al., 1992; Jung, et al., 1996). The function of APP and the products of its proteolytic processing in normal, healthy cells are not yet completely understood. Viable knockout mice strains lacking the APP gene and its homologues APLP1 and APLP2 have been created. However, the offspring of double knockout mice lacking APLP2 and either APLP1 or APP suffer early postnatal lethality, indicating a functionally redundant, yet essential role for the APP family (Heber, et al., 2000; von Koch, et al., 1997). APP knockout mice exhibited reduced locomotor activity and forelimb grip strength, later developing reactive gliosis. Cultured neurons from APP knockout mice also showed dysregulated axonal outgrowth compared to wild-type cells (Perez, et al., 1997). These phenotypes all suggest a role for APP in normal neural function, particularly with regard to neurite outgrowth.
1.2.4: Proteolytic processing of APP and production of amyloid beta The majority of APP in healthy tissue is processed by members of a family of proteases termed -secretases (reviewed in Wolfe and Guenette, 2007). The proteolytic processing of APP is summarised in Figure 1.2. APP may be initially cleaved by an -secretase, which is a member of a disintegrin and metalloproteinase (ADAM) family. Three -secretase candidates have been identified to date; these are ADAM9, ADAM10 or ADAM17; these enzymes cleave APP at a point 83 amino acids from the C-terminus. This cleavage releases the large N-terminal ectodomain of APP (sAPP ) into the extracellular environment, and leaves the shorter C-terminal fragment (C83) embedded in the plasma membrane. The C83 fragment may be subsequently processed by -secretase, a multi-subunit protease complex. 4 Although not completely defined, the minimal components required for an active - secretase are the proteins presenilin (encoded by PSEN1 or PSEN2), nicastrin (NCTH), anterior pharyx-defective 1 (APH-1) and presenilin-enhancer 2 (PEN-2). The proteolytic activity of -secretase is catalyzed by the PS subunit, which is an aspartyl protease. The sAPP and p3 products of the sequential processing of APP by - and -secretases are soluble and non-amyloidogenic. As the recognition site of -secretase occurs within the A region of APP, -secretase processing of APP precludes production of the A peptide. This pathway is often referred to as the non-amyloidogenic pathway of APP processing.
Alternatively, the initial proteolytic cleavage of APP may be performed by another member of the secretase family, -secretase. -secretase, also referred to as -site APP-cleaving enzyme 1 (BACE1), is a type I integral membrane protein which cleaves APP 99 amino acids from its C-terminus. This cleavage releases a slightly smaller APP ectodomain into the extracellular environment (sAPP ), leaving a larger C-terminal fragment (C99) embedded in the membrane. The newly created N-terminal amino acid of C99 corresponds to the N-terminus of A . C99 is then processed by the -secretase complex to produce A . The cleavage site recognized by -secretase in C99 is variable, ranging between residue 38 to 43. While this variable cleavage leads to the production of a heteregenous population of A peptides, in healthy tissues the predominant species produced is 40 amino acids long (A 1-40). A smaller proportion (approximately 10%) is of the longer, more hydrophobic 42 amino acid species (A 1-42). A 1-42 has a much higher tendency to aggregate versus A 1-40, and is the form of A that is the major component of the extracellular plaques found in the brains of Alzheimer's Disease patients. Processing of APP via the and - secretases is referred to as the amyloidogenic pathway.
5
Figure 1.2: Proteolytic processing of the amyloid precursor protein (APP). Proteolytic cleavage of APP by -secretase in the non-amyloidogenic pathway precludes the production of A . However, a small proportion of APP may be initially processed by -secretase, leading to production of the A peptide (Section 1.2.4)
1.2.5: The amyloid cascade hypothesis of Alzheimer's Disease The discovery that the A peptide was a major component of senile plaques suggested a role for the peptide in the pathogenesis of AD. This suggestion focused attention on the APP gene itself, and several lines of genetic evidence now link A and APP to the development of AD. Genetic analysis of families and individuals suffering from hereditary cerebrovascular amyloidosis (Levy, et al., 1990) and early onset and familial forms of AD (Chartier-Harlin, et al., 1991; Goate, et al., 1991; Hendriks, et al., 1992; Mullan, et al., 1992) revealed the presence of a number of mutations in the APP gene. APP mutations have been discovered both within the A region, and externally to it. Mutations occurring
6 within the A coding region result in differing pathologies. Examples include an increased occurrence of intracerebral heamorrhages (due to massive amyloid accumulation in cerebral blood vessels) in the case of the "Dutch" E693Q mutation (Levy, et al., 1990), and early- onset AD with the "Arctic" E693G mutation (due to an enhanced rate of A protofibril formation) (Nilsberth, et al., 2001). The "Swedish" APP mutation occurs outside the A coding region and results in the production of higher amounts of A (Haass, et al., 1995), by reducing the availability of APP to -secretase cleavage, thus increasing flux through the amyloidogenic pathway (Sahlin, et al., 2007).
Mutations have also been discovered in the components of the secretase complexes. Studies on patients suffering from familial AD with raised plasma levels of the A 1-42 peptide revealed mutations in the PSEN1 and PSEN2 presenilin genes, as well as confirming the presence of a number of mutations in APP itself (Scheuner, et al., 1996). These mutations are associated with increased production of A 1-42, an increased proportion of A 1-42 versus A 1-40, or enhanced aggregation of the A 1-42 peptide. Mutations in the PSEN1 and PSEN2 presenilin genes, which form the active proteolytic component of the -secretase complex, also lead to altered APP processing and the generation of a higher ratio of A 1-42 versus A 1-40.
A number of transgenic mice models of Alzheimer's Disease provide additional evidence in support of a central role for the A in the development of the disease. Mice strains bearing the P301L mutant of the human tau gene, a form associated with early-onset AD, display significantly increased formation of neurofibrillary tangles after injection of synthetic A 1- 42 peptide directly into the brain (Götz, et al., 2001) or coexpression of a mutant human APP gene (Lewis, et al., 2001). These transgenic mice models were later extended with the construction of the triple transgenic mouse (3xTg-AD), which in addition to mutant human tau and APP genes, bears an early-onset mutant form of the human presenilin 1 gene (Oddo, et al., 2003). The 3xTg-AD mouse displays a number of features which are consistent with those observed in human AD patients, such as loss of long-term synaptic plasticity, and the appearance of extracellular amyloid plaques and intracellular tau tangles in an age-related manner. It is important to note that in the 3xTg-AD model, deposition of
7 amyloid occurs well before the appearance of tau tangles, despite both APP and tau genes being expressed at comparable levels (Oddo, et al., 2003). Taken together, these transgenic mice models support an upstream role for A in the appearance and progression of hallmark AD features.
Compelling evidence for the key involvement of A in the pathogenesis has also come from anti-A immunization trials in model animals. Immunization of APP transgenic mice protected them from cognitive decline (Janus, et al., 2000; Morgan, et al., 2000; Dodart, et al., 2002), and achieved a similar result in a limited human trial (Hock, et al., 2003). Critically, disappearance of tau pathology (Section 1.2.2) in 3xTg-AD mice occurred after the clearance of extracellular and intracellular A deposits, and was re-detected only after the re-appearance of A deposits when the immunization regimen was halted for an extended interval (Oddo, et al., 2004). These findings demonstrate that tau pathology, such as the formation of NFTs, is likely to be a downstream event of the accumulation of A .
Thus, cumulative evidence indicating a central role for A in the pathogenesis of AD has led to the proposal of the "amyloid-cascade" hypothesis (Selkoe, 1991; Hardy and Higgins, 1992). This hypothesis states that the complex sequence of molecular, cellular and physiological events which eventually leads to onset and progression of fully developed AD is the product of events initiated by the A peptide . The amyloid cascade hypothesis has undergone constant refinement in response to criticisms and to incorporate emerging experimental evidence. These refinements are discussed in more detail below.
1.2.6: Refining the amyloid hypothesis: soluble A oligomers versus plaques Early attention was focused on the appearance of extracellular amyloid plaques as being the causative agent of the neuronal loss in AD. However, there is a poor correlation between postmortem plaque counts and psychometric measures of dementia and mental function assessed ante mortem. (Terry, et al., 1991). Conversely, significant amyloid deposits can be found in the brains of subjects showing no clinical signs of dementia (Lue, et al., 1999). Such evidence precludes a causal role for insoluble amyloid plaques in AD. Alternatively, strong correlations have been detected between the levels of a soluble, oligomeric form of
8 A and clinical markers of AD progression, such as synaptic loss (Lue, et al., 1999) and cognitive decline (Näslund, et al., 2000). More recent studies using ultra-high sensitivity nanoparticle-based detection systems have confirmed that levels of soluble A in cerebrospinal fluid samples are significantly higher from patients diagnosed with AD (via standardized neuropathological tests), versus healthy age-matched controls (Georganopoulou, et al., 2005).
A diverse range of soluble and insoluble A assembly states have been described in the literature, ranging from monomers and so-called low-n oligomers (dimers and trimers), through to larger soluble oligomers (such as dodecamers) and protofibrils, fibrils and the classical insoluble extracellular plaques (reviewed in Haass and Selkoe, 2007). There is still debate as to which of these A assembly states, especially those which are soluble, represent the primary pathogenic agent in AD. SDS-stable dimeric and trimeric forms of A have been described in cell culture models, occurring both intracellularly and secreted into the growth media (Podlisny, et al., 1995; Walsh, et al., 2000). Similar low-n oligomers have also been detected prior to tau NFT formation in CA1 hippocampal samples (Funato, et al., 1999). This is of particular note, as the accepted temporal ordering of senile plaque formation followed by the appearance of tau NFTs is normally reversed in this area of the brain. The detection of small SDS-stable A oligomers before the formation of NFTs provides further support for the amyloid cascade hypothesis.
Recent studies have identified a soluble dodecameric assembly of A with a molecular mass of 56 kDa (referred to as A *56) as a possible pathogenic candidate. In a transgenic mouse model expressing a mutant human APP, concentrations of A *56 correlated inversely with memory performance more so than any other A assembly state examined. Furthermore, A *56 was purified from transgenic mice brain samples, and administered via cannula into the brains of young rats, and this treatment reduced memory performance in standardized tests of spatial memory (Lesné, et al., 2006). Soluble A *56 levels were also inversely correlated more strongly with memory and cognitive performance versus plaque load in transgenic mice (Cheng, et al., 2007). That some mouse strains showed high plaque loads, but performed at normal levels in cognitive tests further reinforces the importance of
9 soluble A species in AD progression, and that the appearance of plaques may even represent a defense mechanism, sequestering pathogenic A oligomers into relatively non- toxic aggregated deposits.
1.2.7: Intracellular and extracellular pools of A In addition to the extracellular pools of A , which include insoluble plaques and fibrils, as well as soluble monomers and oligomers, A may also be found intracellularly. The authenticity of early identifications of intracellular A could be doubted as being false positives due to the potential for antibody cross-reactivity with intact or partially processed APP. However, the development of monoclonal antibodies which specifically recognize the proteolytically processed N- and C-termini of A 1-40 and A 1-42 has resolved any ambiguity regarding the existence of intracellular A peptides (Gouras, et al., 2000; Takahashi, et al., 2002). There is now a substantial body of reliable experimental evidence to support the existence of intracellular A in human brains and several experimental models (reviewed in Laferla, et al., 2007).
A number of studies have proposed a primary role for intracellular A in the development of AD. Intracellular A is detected in the post mortem brains of Down's Syndrome patients many years (decades, in some cases) before the appearance of extracellular A deposits in older patients (Gyure, et al., 2001). The primacy of intracellular A appearance is recapitulated in commonly used transgenic mouse models of AD. Intracellular A is detected prior to the appearance of extracellular A deposits in mice expressing mutant APP and presenilin (Wirths, et al., 2001). Intraneuronal A accumulation also precedes the appearance of tau pathology in 3xTg-AD mice (Oddo, et al., 2003). Results from this study also suggest that intraneuronal A may be the species responsible for the appearance of AD-related phenotypes. Levels of intraneuronal A in 3xTg-AD mice were correlated with the severity of synaptic plasticity defects, and the development of these defects preceded the appearance of both senile plaques and NFTs (Oddo, et al., 2003).
Intracellular A has been detected in various areas of the cell, including compartments of the endosomal/lysosomal system (Koo and Squazzo, 1994), multivesicular bodies (MVB)
10 (Takahashi, et al., 2002) and the organelles of the secretory pathway (Busciglio, et al., 1993). Some of these locations, such as the ER/Golgi, may reflect sites of intracellular A production. Others, such as the endosomes and MVBs may indicate uptake from the extracellular environment. The accumulation of A in MVBs has been shown to be deleterious, resulting in disrupted MVB sorting and proteasomal inhibition (Almeida, et al., 2006). Proteasomal inhibition is also observed in other models of intracellular A accumlation such as 3xTg-AD mice (Tseng, et al., 2008), suggesting a mechanism through which intracellular A leads to downstream pathologies. As the proteasome is located in the cytosol, it also implies there may be a cytosolic pool of A , possibly destined for destruction by the proteasome (Laferla, et al., 2007).
1.3: Parkinson's Disease Parkinson's Disease (PD), named after James Parkinson who first described the symptoms of the disease in 1817, is the most commonly occurring neurodegenerative disease affecting movement. The disease has an incidence of approximately 1% in persons over 60 years of age, and is superceded only by AD as the most common neurodegenerative disease overall. As with AD, the prevalence of PD increases significantly with age, rising from 0.6% for 65 year olds to 3.5% for 89 year olds (Fahn and Sulzer, 2004). This number does not include the large population of asymptomatic sufferers who may be undiagnosed, as physical symptoms of PD are not apparent until 70-80% loss of the dopaminergic neurons has occurred (Schapira, 1999).
1.3.1: Clinical symptoms of Parkinson's Disease The physical symptoms of PD are characterized by a number of deficits of motor control, including a resting tremor, shuffling gait, rigid mask-like facial expressions, bradykinesia (slowness of movement), and akinesia (lack of ability to initiate movement). In many instances there may also be non-physical symptoms, such as cognitive, sensory and sleep disturbances. These appearance of these symptoms may precede diagnosis of PD by years. The clinical symptoms of PD are due to a loss of striatal dopamine, a key neurotransmitter involved in initiating and controlling signaling in motor neurons. This loss is due to the selective loss of dopaminergic neurons from a pigmented region of the midbrain known as the substantia nigra pars compacta (reviewed in Fahn and Sulzer, 2004).
11 1.3.2: alpha-synuclein is the major component of Lewy Bodies A prominent histological feature present in neurons of the substantia nigra from PD patients are the spherical, cytoplasmic proteinaceous aggregates known as Lewy Bodies (LBs). As with the extracellular amyloid plaques found in the brains of AD patients (Section 1.2.3), it is not known whether Lewy Bodies represent a toxic entity themselves, or are the endpoint of cellular efforts to sequester and detoxify other deleterious protein species. Lewy Bodies are heterogenous in composition, and are heavily polyubiquitinated (Kuzuhara, et al., 1988; Iwatsubo, et al., 1996), hinting at the involvement of the proteasomal degradation pathway in PD.
A major advance in the understanding of PD at a molecular level came with the discovery via immunohistochemistry that the protein -synuclein was the major component of LBs (Spillantini, et al., 1997). -Synuclein is a 140 amino acid protein normally localized to the synaptic terminals of neurons. The role of -synuclein in healthy tissue is still uncertain. Deletion of the -synuclein homologue in mice is non-lethal, and mutant mice exhibit wild- type neuroanatomy and dopaminergic release and re-uptake in response to simple stimuli (Abeliovich, et al., 2000). However, altered dopamine release in response to multiple stimuli and amphetamine administration was observed, suggesting a role for -synuclein in the activity-dependent regulation of presynaptic dopamine pools. This is reinforced by findings that knockdown of -synuclein expression in neural cell culture results in depletion of the distal pool of presynaptic vesicles (Murphy, et al., 2000). The biophysical properties of -synuclein, such as its propensity for membrane association and the changes in its structure which are induced when this occurs (Davidson, et al., 1998; Perrin, et al., 2000), involvement in lipid metabolism (Sharon, et al., 2003; Golovko, et al., 2005) correlate with these proposed functions.
Immunohistochemical data hinting at the involvement of -synuclein in PD is strongly supported by genetic evidence. A number of point mutants of -synuclein have been identified which led to early-onset PD, including A53T (Polymeropoulos, et al., 1997), A30P (Krüger, et al., 1998) and E46K (Zarranz, et al., 2004). Increases in -synuclein copy number led to an earlier age of onset of PD. Three copies of the -synuclein gene lead to
12 appearance of PD symptoms in the fifth decade of life (Chartier-Harlin, et al., 2004); four copies of -synuclein result in appearance in the third decade (Singleton, et al., 2003). The deleterious effects of high -synuclein gene dosage are recapitulated in animal models. Targeted overexpression of human -synuclein in the dopaminergic neurons of the substantia nigra of mice resulted in the appearance of cytoplasmic, -synuclein-positive inclusions, selective dopaminergic cell death, decreases in striatal dopamine, and development of motor impairment (Kirik, et al., 2002).
-Synuclein has a high propensity towards self-aggregation. -Synuclein aggregates may be observed in many states, ranging from soluble monomers, "reversible" oligomers (which may revert to monomers) (Cole, et al., 2002), oligomers which display amyloid-like structural characteristics (Conway, et al., 1998; Conway, et al., 2000), fibrils (Wood, et al., 1999), and large mature LBs. There is ongoing debate as to which assembly state represents the species responsible for the toxic effects of -synuclein. As with amyloid plaques in A , there has been a shift from considering the large insoluble LBs as being pathogenic agents. Rather, LBs may represent a protective response by the cell to sequester otherwise toxic -synuclein oligomers and fibrils into an inaccessible, insoluble state (Kramer and Schulz-Schaeffer, 2007; Robinson, 2008).
1.3.3: Environmental factors and Parkinson's Disease Despite the identification of multiple PD-associated loci (reviewed in Thomas and Beal, 2007), fewer than 10% of PD cases can be attributed to heritable mutation. The remaining cases have no known etiology, and are referred to as sporadic or idiopathic PD. An important advance came with the discovery of acute PD-like symptoms in a small group of intravenous users of a new "synthetic heroin", 1-methyl-4-phenyl-4-propionoxy-piperidine (MPPP). These patients began to develop classical PD symptoms, such as uncontrolled movement of limbs, stiffness, fixed stare and a shuffling gait within a week of using the drug. As in sporadic cases of PD, these symptoms could be alleviated by the administration of L-dopa; symptoms returned after the cessation of L-dopa treatment, and showed no signs of remission five months after onset (Langston, et al., 1983). Post-mortem examination revealed selective degeneration of the substantia nigra (Davis, et al., 1979). Analysis of
13 drug samples revealed they were contaminated with 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP), an unintended byproduct of MPPP synthesis. MPTP is selectively transported into dopaminergic cells, where it is converted by monoamine oxidases into the cation 1-methyl-4-phenylpyridinium (MPP+) (Langston, et al., 1984), a potent inhibitor of complex I of the mitochondrial electron transport chain; it is by this mechanism that MPTP is thought to exert its toxicity.
The chemical structure of MPP+ is very similar to that of paraquat, a widely used herbicide (Figure 1.3). This similarity, combined with the severe Parkinsonian effects of MPTP administration in humans and primates, led to the hypothesis that environmental agents such as pesticides may play an important role in the development of PD. A number of epidemiological studies of PD provide evidence to support this hypothesis (reviewed in Cory-Slechta, et al., 2005). The incidence of PD is higher in more heavily industrialized nations, and this association is preserved even where genetic variations of particular populations is taken into account. The incidence of idiopathic PD is higher in US citizens of either Japanese/Okinawan or African ancestry, when compared with age and sex matched populations in Asian countries or Nigeria, respectively (Morens, et al., 1996; Schoenberg, et al., 1988). There are also regional differences in the incidence of PD within individual nations, suggestive of localized environmental influences on the appearance of idiopathic PD cases (Lanska, 1997; Svenson, 1990). The environmental hypothesis is further bolstered by studies which examined the incidence of PD in monozygotic versus dizygotic twins (Tanner, et al., 1999) and in twins versus the general population (Wirdefeldt, et al., 2008). In these studies, the prevalence of PD in monozygotic twins was comparable to that of the respective comparison populations, suggesting that in many cases, factors other than genetics plays a key role in the development of the disease.
14
Figure 1.3: Chemical structures of MPTP, MPP+ and paraquat
Extensive epidemiological studies have uncovered a wide range of factors which may be associated with a higher risk of developing PD. These include living in an industrialized country, living on a farm, consuming well water, working with or exposure to herbicides (Brighina, et al., 2008), insecticides, fungicides, and certain metals such as lead, manganese, copper and iron (reviewed in Brown, et al., 2006; Elbaz and Tranchant, 2007 ;Elbaz and Moisan, 2008).
These analyses have also revealed environmental factors which may be protective against the development of PD, such as consumption of coffee or black tea (Tan, et al., 2008; Hu, et al., 2007) and higher levels of plasma uric acid (ie: gout) (Weisskopf, et al., 2007). Surprisingly, cigarette smoking also appears to provide significant protection against onset of PD (Ritz, et al., 2007; Thacker, et al., 2007). Farnesol, an additive to cured tobacco, may be responsible for this protective effect via inhibition of monoamine oxidase, thus reducing the turnover of dopamine (Khalil, et al, 2006).
15 1.3.4: Pesticides and Parkinson’s Disease Pesticide exposure has been consistently identified as a risk factor associated with the incidence of PD from epidemiological studies, with extensive biological evidence providing potential mechanistic explanations (reviewed in Brown, et al., 2006; Hatcher, et al., 2008). The term "pesticide" encompasses a chemically diverse family of compounds with a wide range of applications, including herbicides, insecticides, rodenticides and fungicides. This diversity is reflected in the fact that only specific classes of compounds have been demonstrated to have a strong association with PD via epidemiological analysis or toxicological experiments, despite the nearly ubiquitous presence of pesticides in the modern environment. A selection of these compounds is dicussed in more detail below.
1.3.5: Paraquat As mentioned in Section 1.3.3, paraquat is structurally similar to the archetypal Parkinsonian neurotoxin MPP+, and has thus been extensively scrutinized by PD researchers. Paraquat is used commercially as a non-selective contact herbicide, capable of destroying plant tissue upon contact (Corasaniti, et al., 1998). Although banned in many Western countries, paraquat is still commonly deployed in less developed nations. The acute toxicity of paraquat is largely due to its ability to participate in redox cycling reactions which lead to the production of the superoxide radical, and also the regeneration of the paraquat molecule itself. This redox cycling also results in the depletion of cellular stores of glutathione and NADPH (Bus and Gibson, 1984). Although the primary effect of acute paraquat exposure in humans is severe pulmonary toxicity, brain damage has also been detected in those receiving a lethal dose (Hughes, 1988). Case-controlled epidemiological studies in Taiwanese populations have determined that individuals with over 20 years of occupational exposure to paraquat experience an increased risk of developing PD (Liou, et al., 1997).
Studies in model systems using paraquat have resulted in the re-creation of a number of PD-like pathologies. In mice, systemic exposure to paraquat via intraperitoneal injections led to the highly selective loss of dopaminergic neurons in the substantia nigra. However, overall striatal dopamine levels were not significantly reduced, which may be due to a compensatory response by remaining neurons (McCormack, et al., 2002). Expression of -
16 synuclein in mice was increased after repeated injections of paraquat, with accompanying accumulation of -synuclein-rich aggregates in the neurons of the substantia nigra (Manning-Bog, et al., 2002), and this is a key Parkinsonian pathology which is not reproduced in MPTP/MPP+ toxicant models. Intriguingly, overexpression of human - synuclein in a transgenic mouse model prior to paraquat treatment resulted in a neuroprotective effect (Manning-Bog, et al., 2003). This stands in contrast to the protective effect of -synuclein deletion against MPTP-induced dopaminergic cell death in mice (Dauer, et al., 2002), and hints at a complex relationship between -synuclein and environmental toxins.
The most readily apparent mechanistic explanation for the neurotoxicity of paraquat may be its participation in redox cycling and generation of ROS. Pre-treatment of rat dopaminergic cell cultures and adult mice with a synthetic superoxide dismutase/catalase mimetic prior to paraquat exposure prevented dopaminergic cell death (Peng, et al., 2005). Similarly, transgenic mice overexpressing superoxide dismutase or glutathione peroxidase were more resistant to the dopaminergic cell death resulting from paraquat treatment (Thiruchelvam, et al., 2005). In addition to ROS-related toxicity, lower expression levels of the 19S proteasomal subunit, proteasomal dysfunction, and increased intracellular accumulation of -synuclein aggregates were reported in human neuroblastoma cell cultures after treatment with paraquat (Yang and Tiffany-Castiglioni, 2007).
1.3.6: Rotenone Rotenone is a highly hydrophobic compound derived from the roots of a number of species of tropical plants. It is used commercially in the management of nuisance fish populations in ponds and lakes, and due to its botanical source, it is also widely utilized in organic farming as a pesticide. Rotenone's hydrophobic properties allow it to easily cross the blood-brain barrier and penetrate cellular membranes. In the mitochondria, rotenone binds to and inhibits complex I of the electron transport chain (ETC), blocking the normal flux of electrons, and leading to the production of ROS via electron leakage from components of the ETC upstream of complex I (Hensley, et al., 1998). As complex I deficiencies exist in the brains of PD patients (Schapira, et al., 1989), and MPP+ related neurotoxicity is
17 thought to be mediated via the same mechanism, rotenone models of PD have been developed.
Systemic administration of rotenone in rats results in the appearance of a number of key features of PD pathology, including the selective degeneration of nigrostriatal dopaminergic neurons (despite rotenone-mediated complex I inhibition occurring throughout the entire brain), appearance of intraneuronal ubiquitin and -synuclein- positive aggregates, and development of motor defects (Betarbet, et al., 2000; Alam and Schmidt, 2002; Sherer, et al., 2003). This inhibition of complex I in rotenone-treated rats also leads to extensive oxidative damage to brain tissue in similar regions to those affected in the brains of authentic PD patients (Sherer, et al., 2003). Human neuroblastoma lines expressing the rotenone-insensitive Saccharomyces NADH dehydrogenase NDI1 gene (which can substitute for some aspects of human complex I function) were resistant to the toxic effects of rotenone, and did not develop -synuclein-rich cytoplasmic inclusions. Pre-treatment of cultured rat mid-brain slices with the anti-oxidant -tocopherol (vitamin E) also prevented dopaminergic cell death associated with rotenone treatment (Sherer, et al., 2003). These results demonstrate an important role for complex I dysfunction and ROS in PD pathology, and indicate that antioxidant-based therapeutic approaches to the treatment of PD may hold promise.
Despite evidence for an involvement of rotenone in the development of PD, there have been no significant epidemiological associations detected between rotenone exposure and the onset of PD in humans. Unlike the highly persistent organochlorines, rotenone is relatively unstable in the environment (half-life approximately 0.1 years), and has not been as widely utilized, which may help explain the lack of association. However, the strength of existing toxicological and biological data on rotenone may portend an as-yet undiscovered role in PD development of other compounds which also act as mitochondrial complex I inhibitors.
1.3.7: Organochlorines Organochlorine pesticides, which include compounds such as dichlorodiphenyltrichloroethane (DDT), chlordane, and the cyclodienes dieldrin and aldrin,
18 were heavily utilized from the 1950s through to the 1970s as insecticides, and were applied to a variety of food and non-food crops (Hatcher, et al., 2008). Following the realization that these compounds were linked to conditions such as immune, nervous and reproductive dysfunction, their use was largely restricted in developed nations. However, due to the high stability of organochlorines in the environment (half-life of 5.5 years for DDT; 2.7 years for dieldrin), these compounds have remained near-ubiquitous pollutants decades after their widespread use ceased. Due to the hydrophobicity of cyclodienes such as dieldrin, they also tend to bioaccumulate and concentrate at the top of food chains. To this date, significant quantities of dieldrin are still present in human foodstuffs such as cow’s milk, beef and fish. In the United States, where the use of dieldrin was banned in the late 1970s, it is estimated that in the late 1990s a child may still be exposed to more than the US Environmental Protection Agency's maximum recommended level of the pesticide, via normal food intake (Schafer and Kegley, 2002).
Post-mortem examination of the brains of PD patients has revealed significantly higher concentrations of polychlorinated biphenyls (PCB) (Corrigan, et al., 1998) and the organochlorine pesticide dieldrin (Corrigan, et al., 2000) compared to non-neurological control brains. This significance was maintained even where post-mortem PD brain samples were compared to AD brain samples, indicating the accumulation of dieldrin was specifically associated with PD rather than neurodegenerative disorders in general (Fleming, et al., 1994). In addition to evidence from post-mortem examinations, family- based case-control epidemiological studies have demonstrated a significant association between organochlorine pesticide exposure and incidence of PD (Hancock, et al., 2008).
The toxicological effects of dieldrin on organismal and cellular models have been extensively studied and reviewed (Kanthasamy, et al., 2005). Dieldrin treatment results in the dose-dependent production of ROS in several cell types including murine dopaminergic cells (Chun, et al., 2001), and pre-treatment of cells with SOD or with ROS-scavenging drugs abolished this production (Kitazawa, et al., 2001). Pro-apoptotic signaling and effector pathways are activated after dieldrin exposure. These include release of mitochondrial cytochrome c and stimulation of caspase cleavage (which could be inhibited
19 by treatment with ROS-scavengers) in human PC12 cells (Kitazawa, et al., 2003). Proteasomal function is compromised in neuroblastoma cells upon exposure to low concentrations of dieldrin (Wang, et al., 2006). The severity of proteasomal inhibition also appears to be related to the length of exposure. Short-term dieldrin treatment of a rat dopaminergic cell line expressing human -synuclein led only to an increase in the level of ubiquitinated proteins; prolonged treatment resulted in inhibition of the ubiquitin- proteasome system, and the appearance of -synuclein aggregates (Sun, et al., 2005). Of particular relevance to studies of the relationship between dieldrin and PD are findings that dieldrin treatment results in dopamine depletion and dysregulation of the dopaminergic regulatory systems in mice (Richardson, et al., 2006). This effect is mediated through dieldrin-induced changes in the expression of the dopamine transporter DAT, and vesicular monoamine transporter 2 (VMAT2), two transporters which play a key role in the maintenance of dopamine homeostasis. Dieldrin treatment of this mouse model also resulted in increased sensitivity to additional neurotoxic insults, such as MPTP treatment. Intriguingly, perinatal dieldrin treatment resulted in these effects being more pronounced in male offspring versus female offspring, which reflects the gender-specific bias in PD incidence observed in human populations.
Many studies of dieldrin toxicity have focused on the acute effects of the pesticide, relying on concentrated doses administered over relatively short periods of time. An animal model of chronic environmental dieldrin dosing has also been developed, in which mice are treated with dieldrin at sub-acute concentrations for 30 days (Hatcher, et al., 2007). After this period, the pesticide was found to accumulate in a dose-dependent manner in the brain. This treatment also resulted in the disruption of dopamine metabolism, and a decrease in the expression of striatal dopamine transporters. Markers of oxidative stress and damage, such as protein carbonyl formation and glutathione depletion were significantly increased, as was expression of -synuclein. These findings provide valuable clues as to the mechanisms by which dopaminergic neurons may be compromised by the types of chronic, low-level exposure to pesticides which are hypothesized to be responsible for the onset of PD in many cases.
20 1.3.8: Multiple hit models of Parkinson's Disease Combined, all known PD-associated genetic loci account for only approximately 10% of PD cases, and while extensive epidemiological analysis supports the notion of an environmental contribution towards the development of PD (section 1.3.3), a single causative agent has not been unequivocally identified. This scenario is suggestive of an interplay between multiple, overlapping neuronal insults which eventually give rise to the disease state (Sulzer, 2007; Carvey, et al., 2006). This is analogous to the accepted "multiple-hit" hypothesis of tumour formation, which only occurs after the failure of several layers of cellular control systems (Compagni and Christofori, 2000).
Beyond controlled experimental conditions, exposure to a single homogeneous environmental pollutant is highly unlikely. More realistically, an individual will be simultaneously exposed to a heterogeneous mixture of agents which may exhibit unforeseen interactions with each other or with the individual's genetic predispositions. Indeed, consumption of the typical daily diet in the western United States could be expected to result in exposure to at least five persistent pesticides (Schafer and Kegley, 2002). The homeostatic mechanisms of the brain, particularly the relatively vulnerable dopaminergic neurons, may be able to maintain an effective defense against single genetic or chemical stresses. However, as subsequent stresses mount on these defenses, they may become overwhelmed leading to cellular damage or death. Experimental evidence supports this hypothesis. For example, incubation of purified -synuclein with combinations of pesticides and metals leads to synergistic enhancement of -synuclein fibrillization (Uversky, et al., 2002). In a mouse model, administration of paraquat and maneb (manganese ethylenebisdithiocarbamate, an antifungal compound) individually at low concentrations did not result in a phenotype. However, when these compounds were administered at the same concentration simultaneously their effect was potentiated, and nigrostriatal cell death and development of a PD phenotype was observed (Cory-Slechta, et al., 2005). As in dieldrin-treated mouse models (Richardson, et al., 2006), this phenotype was more severe in male mice than in female mice, again reflecting findings from epidemiological studies.
21 1.4: Saccharomyces as a model organism As alluded to in the preceding sections, a complete molecular understanding of the mechanisms underlying the pathogenesis of AD and PD remains elusive. The development of both diseases is influenced by a comprehensive range of endogenous and exogenous factors, which in turn lead to the disruption and dysregulation of a similarly diverse set of cellular processes. There are obvious ethical and practical constraints on the use of human subjects in neurodegenerative research. Access to living human brain tissue is understandably difficult, the supply of appropriately matched diseased and control post- mortem tissue is limited. The late age of onset in the majority of cases of AD and PD also dictates that longitudinal studies aimed at identifying early events in disease pathogenesis will be difficult.
The baker's yeast Saccharomyces cerevisiae is an important model of eukaryotic cell biology. Analysis of the fully sequenced S. cerevisiae genome has revealed that over 30% of its genes exhibit a high level of homology with Homo sapiens (defined by having a BLASTP e score of less than 1 x 10-10) (Botstein et al, 1997). Genes involved in key eukaryotic cellular processes, such as energetics, regulation of the cell cycle, and DNA repair and replication show especially high degrees of homology between yeast and humans. This degree of homology is conserved when only a subset of human genes known to be associated with disease is considered, with approximately 30% of these genes having yeast homologs. Thus a large number of the biochemical and physiological processes which are disturbed in human disease may be conserved in yeast, providing a convenient platform for their characterization.
In addition to a completely sequenced and highly annotated genome (Goffeau, et al., 1996), the unique genetic characteristics of S. cerevisiae make it especially suitable for large scale genome-wide functional studies, which are the essence of modern integrative approaches to the study of biology. One of these characteristics is the phenomenon of homologous recombination. The ease with which foreign segments of DNA may be incorporated into targeted regions of the S. cerevisiae genome not only facilitates the stable integration of
22 foreign genes into the host chromosome, but also allows for the facile interruption and deletion of genes.
A consortium of laboratories has taken advantage of the technique of gene disruption by homologous recombination of an auxotrophic marker cassette, to systematically disrupt all known ORFs in the S. cerevisiae genome (Winzeler, et al., 1999) The product of this effort is a collection of approximately 4600 mutant strains, representing the complete set of non- lethal, single gene disruptions of the S. cerevisiae genome. The Saccharomyces genome knockout collection is commercially available, with mutant strains being arrayed into approximately 50 standard 96-well microtitre plates. By probing this collection with various experimental treatments (such as stresses, drugs or heterologous gene expression) and assaying the phenotypic “read-out”, it is possible to identify which genes play key roles in the cellular responses to these treatments. The knockout collection has been exploited to identify genetic networks involved in a broad range of cellular processes, including maintenance of genomic integrity (Huang, et al., 2003), response to oxidative stress (Thorpe, et al., 2004) and resistance to anti-cancer drugs (Dilda, et al., 2008). The presentation of the knockout collection in 96-well plate arrays, and the simplicity and flexibility of the agar spot-test for the assessment of growth also makes the collection highly amenable to automation.
In addition to the Saccharomyces gene knockout collections, vector and strain sets have also been created to allow inducible expression of virtually any S. cerevisiae ORF. These sets include ORFs fused with affinity tags to facilitate purification under various conditions (including non-denaturing conditions to allow identification of proteins interacting directly with the purified “bait” protein) (Gavin, et al., 2002) and ORFs fused with GFP, which has not only lead to the determination of the cellular localization of many S. cerevisiae proteins (Huh, et al., 2003), but also to the estimation of their copy number (Ghaemmaghami, et al., 2003). The integration of the results of these genome-wide experiments with other information, such as curated literature references into powerful and convenient bioinformatic resources such as the Saccharomyces Genome Database (http://www.yeastgenome.org), together with the commercial “off the shelf” availability of
23 the vectors and strains makes Saccharomyces a powerful and convenient platform for the study of eukaryotic cell biology.
1.4.1: Yeast models of neurodegeneration Given the benefits of S. cerevisiae and the caveats of existing experimental models of neurodegenerative disorders, it is perhaps unsurprising that there has been a rapid growth in the application of S. cerevisiae to modeling aspects relating to neurodegenerative disorders in recent years. These models are based on the "humanization" of yeast cells, by expression of one of more human genes which are associated with the relevant neurodegenerative pathology. A brief summary of yeast models for the study of neurodegeneration developed to date is presented in Table 1.2. It should be noted that the S. cerevisae genome does not contain homologs for any of the key genes involved in these diseases, thus avoiding potential interference from native genes in the interpretation of experimental observations.
Table 1.2 Summary of published S. cerevisiae Alzhiemer's Disease (AD) and Parkinson's Disease (PD) models. These models are all based on the approach of expression of one of more human disease-associated genes in yeast so as to recapitulate key features of the disease. Table adapted and extended from Winderickx et al (2008).
Disease Protein Key findings Reference
* - $ < ( & 5 #$5 6 ) ) ( = 0 4 ) > = 0 ; " 6 + &<4 07? / - 6 1 " + &<4 07? - # 6 @ $( ., + @ ? 0 $( / ?A ( 6 B ) C 00 ? < ( - -- 24 - 0 -- $( C ? < - 6 ., 6 , D 0 4 - ? 6 ) ( ( 6 0 ( 4 - ?6 ( ,? 4 *'&6 - ( &&< ( 6 ? + 4 ? - -6 , / " 6 ., ( 0 D ( 6! . " 6 ( 6> ? ( 6 '- ( + " * $*. ( 6 < ,? @ - - & B 1?/ 0 6
1.4.2: Yeast models of Parkinson's Disease: -synuclein Table 1.2 illustrates the substantial and rapidly evolving contributions which have come from yeast models of neurodegeneration, particularly with respect to -synuclein and PD. The initial discovery by Willingham et al (2003) that genes relating to lipid synthesis and vesicular trafficking played critical roles in the cellular response to -synuclein proved to be prescient. Subsequent studies have demonstrated that -synuclein was trafficked via the secretory pathway to the plasma membrane (Dixon, et al., 2005), inhibited the activity of
25 lipid remodeling enzymes and increased accumulation of lipid droplets (Outeiro and Lindquist, 2003), interfered with endocytosis (Zabrocki, et al., 2005), and that its toxic effects were intimately linked to its association with lipid (Brandis, et al., 2006). This experimental track has recently lead to the discovery that at least one mechanism by which -synuclein exerts its toxicity is via inhibition of ER to Golgi vesicular trafficking (Cooper, et al., 2006). The authors detailed not only the discovery of a human gene which ameliorated -synuclein-related toxicity (Rab1), but also the successful translation of this finding to animal and mammalian cell culture models of PD. Some authors of this report have also described the use of a yeast -synuclein model for high throughput screening a library of over 350,000 compounds which may alleviate the -synuclein toxicity. A number of compounds have successfully been identified and their beneficial effects have been confirmed using C. elegans and neuronal cell culture models of PD (Fleming, et al., 2008). Although the identity of these compounds is not disclosed, a suggestion is made that these may target the same ER-Golgi vesicular trafficking pathway as Rab1.
The contribution of the S. cerevisiae PD model is not limited to examining the effects of - synuclein on lipid metabolism and vesicular trafficking. As reviewed in Section 1.3.3, oxidative stress, apoptosis and environmental factors, such as exposure to metals, are all implicated in the pathogenesis of PD. Expression of -synuclein in yeast sensitizes cells to exposure to elevated levels of iron, zinc or hydrogen peroxide (Zabrocki, et al., 2005; Griffioen, et al., 2006; Flower, et al., 2005). This sensitivity may reflect the increased levels of endogenous ROS that accompany -synuclein expression in yeast, and that supplementation of growth media with the reductant glutathione relieves -synuclein toxicity (Flower, et al., 2005). These authors also determined that a mild heat shock prior to the induction of -synuclein expression, treatment with geldanamycin (a drug which induces the heat shock response), and overexpression of the HSP70 family member Ssa3p all resulted in the relief of -synuclein toxicity.
1.4.3: Yeast models of Alzheimer's Disease: APP, tau and A While groups developing yeast as a PD model have largely focused on the role of a single disease-associated protein, -synuclein, a wider range of genes related to AD have been
26 examined (summarized in Table 1.2). A notable example was the use of S. cerevisiae as a cellular host for the reconstitution of -secretase activity (Edbauer, et al., 2003). In this study, four human genes were coexpressed, encoding presenilin-1 (PS1), nicastrin (Nct), anterior pharynx-defective 1 (APH-1), presenilin enhancer 2 (PEN-2). In addition, a fragment of APP fused to a GAL4 reporter was co-expressed to detect -secretase activity. Correct in vivo scission of the APP fragment was detected, and analysis of a membrane protein extract from the humanized cells showed that formation of an intact -secretase complex had occurred, and this complex was able to process full length APP in an in vitro assay. In this case, the lack of yeast homologs to the human -secretase genes allowed an in vivo reconstruction of a large multi-enzyme complex, without interference from native genes which may have confounded interpretation.
Tau, a microtubule binding protein responsible for the formation of the characteristic intracellular neurofibrillary tangles in AD has also been expressed in yeast (Vandebroek, et al., 2005). Expression of tau alone was not reported to result in toxicity. However, analysis of purified tau from yeast extracts revealed recapitulation of a number of AD relevant features. These included phosphorylation of tau at residues known to be hyperphosphorylated in AD, and the identification of the yeast protein Mds1p, a homolog of mammalian GSK-3 , as the kinase responsible for this phosphorylation. Additionally, hyperphosphorylated tau formed fibrils in vitro, whereas unphosphorylated tau purified from MDS1 mutants did not. Finally, antibodies which recognize tau conformations associated with AD pathology in humans were also able to recognize tau purified from the yeast expression model. Therefore, a S. cerevisiae tau expression system could prove to be a convenient and pathologically relevant source of tau for further characterization, as well as a potential cellular model for elucidating pathways involved in toxicity.
More recently, S. cerevisiae models expressing the A peptide have been developed. These models differ from the type described by Edbauer et al (2003) in that no attempt is made to reconstitute a complete secretase pathway together with APP expression and processing. Rather, expression constructs corresponding only to the A domain of APP are used, as the focus of these models is the study of A aggregation in an intracellular environment. As
27 reviewed in Section 1.2.7, intracellular A is believed to play a potentially causative role in the early progression of AD, and an understanding of the assembly of A in this environment into oligomers or insoluble aggregates is highly desirable. Two groups have independently described an elegant assay for A oligomerization based on changes in colony colour, which is achieved by exploiting the unique properties of yeast Sup35p, a transcriptional terminator and naturally occurring prion (Bagriantsev and Liebman, 2006; von der Haar, et al., 2007). Briefly, the N-terminal prion forming domain of Sup35p is substituted with A . A mediated aggregation of the A -Sup35p fusion protein results in loss of Sup35p transcriptional termination function. This loss of function results in transcriptional read-through of a nonsense codon in a mutant ade1 gene and the accumulation a red coloured intermediate in the adenine biosynthetic pathway. The subsequent appearance of red or white colonies provides a convenient indication of A aggregation. This approach is discussed in further detail in Section 3.3.1.2.
Using this approach, both groups reported that A 1-42 forms oligomers and aggregates when expressed in the cytosol of S. cerevisiae. Expression of A 1-40 and less amyloidogenic point mutant A 1-42 Sup35p fusions resulted in the reduction or abolition of aggregate formation, accurately reproducing the known behaviour of these peptides in AD and mammalian AD models. Of particular note was the involvement of yeast chaperone Hsp104p in the formation of A oligomers. Hsp104p is involved in shearing of insoluble prion aggregates (Kryndushkin, et al., 2003), bound directly to A 1-42-Sup35p, and appears to be necessary for the oligomerisation of the fusion protein in S. cerevisiae. Additionally, the prion curing agent guanidine was found to accelerate oligomerisation of A (Bagriantsev and Liebman, 2006). The identification of genetic and chemical factors which affect A oligomerisation may lead to the discovery of homologs and conserved pathways in humans, and illustrates the value of the S. cerevisiae intracellular A model.
The direct visualization of intracellular A aggregation and localization in S. cerevisiae has also been investigated, via the expression of an A 1-42-GFP fusion protein (Caine, et al., 2007). In wild-type cells, expression of the A 1-42-GFP fusion lead to the formation of
28 large fluorescent puncta in the cytosol which appeared as amorphous patches when visualized using immunoelectron microscopy, possibly corresponding to the formation of intracellular aggregates or inclusions. Although expression of the A 1-42-GFP fusion resulted in only a mild reduction in growth, a pronounced induction of the heat shock response occurred. Taken together with the involvement of Hsp104p in A -Sup35p oligomerisation, these results indicate a role for heat shock proteins and chaperones in the response of the cell to the accumulation of intracellular A , and support findings that levels of heat shock proteins are increased in the brains of AD patients (Perez, et al., 1991) and in cellular models expressing intracellular A (Magrané, et al., 2004).
1.5: Aims of this study While significant advances have been made in elucidating the molecular mechanisms of AD and PD, key questions remain to be answered. What factors affect the production, misfolding and assembly of intracellular A into higher order structures? What are the precise intracellular locations A ? How does intracellular A lead to toxicity and cell death? How do the effects of environmental insults overlay with the deleterious effects of -synuclein overexpression and mutation? The development of experimental systems which combine tractability with the recapitulation of fundamental molecular processes in AD and PD to complement existing models is worthy of serious attention.
Therefore, the aims of this study were
• The development and validation of a yeast model for the study of intracellular A aggregation and localization, which is amenable to large high-throughput screening studies.
• The exploitation of this model for the discovery of factors affecting the aggregation and localization of intracellular A , by screening the Saccharomyces gene knockout collection.
29 • The application of a yeast model of -synuclein pathobiology to study the and to explore interactions between genetic factors (such as early-onset PD associated - synuclein mutations) with suspected environmental risk factors.
30
Chapter 2: Materials and Methods
2.1: General materials The source of material not otherwise noted in the text are summarised in Table 2.1. General chemicals were purchased from Sigma Aldrich, BDH, or Ajax Chemicals and unless otherwise stated were of analytical grade.
Table 2.1: Materials and reagents
Material Supplier " & 95&C: 9!: '( 95&C: 95 : ! 95&C: 9 : & 95&C: / '( 95&C: & 95&C: & 95&C: & 95&C: $ & 95&C: & 9 & 95&C: : 9$.<<4 : & 95&C: ) #+ % 095&C: 8 ( '( 95&C: 8 9? 95&C: : 5 !- ) '( 95&C: E & 95&C: + & 95&C: ) & 95&C:
2.2: Escherichia coli media, strains, selection and storage E. coli strains were cultured in Luria-Bertani (LB) medium (10 g/l tryptone, 5 g/l yeast extract, 5 g/l sodium chloride) at 37oC.
Media was supplemented with antibiotics as appropriate for selection and maintenance of cells harbouring plasmids. Antibiotics were stored as concentrated stocks (1000x) at
31 -20oC, and used at the following final concentrations: ampicillin 100 μg/ml; chloramphenicol 50 μg/ml; tetracycline 20 μg/ml; kanamycin sulphate 50 μg/ml.
For general molecular biology applications, either XL-10 Gold (Stratagene; Tetr (mcrA)183 (mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F´ proAB lacIqZ M15 Tn10 (Tetr) Amy Camr]) or DH5 (Invitrogen; F- recA1 endA1 hsdR17(rk-, mk+) supE44 - thi-1 gyrA96 relA1) cells were used.
E. coli strains were prepared for long-term storage by pelleting and removal of growth media, resuspension in sterile aqueous 50% (v/v) glycerol, and storage at -80oC.
2.3: S. cerevisiae media and storage Yeast strains were propagated and maintained on YEPD medium (2% w/v glucose, 2% w/v bacteriological peptone, 1% w/v yeast extract) at 30oC. In general experiments were performed in synthetic complete (SC) media (0.5% ammonium sulphate, 0.17% yeast nitrogen base) (containing amino acids and other supplements as indicated in Table 2.2). The carbon source used in SC media was either 2% (w/v) glucose or galactose, depending on whether galactose induction of a gene of interest was desired. SC media also contained supplements as listed below in Table 2.2.
Table 2.2: Supplements used in synthetic complete (SC) media
Component Concentration (μg/mL) B 1 @" 1 @" 1 @" 1 @" 1+ @" 1. @" 1. @" 1. @" 1# @" 1! @" 11 " 11 @"
32 1% <@" B 1 @" 1 @" 1& @" 1) @" 1) @" 1) @" > @" 1= @"
1: Not added where histidine (HIS3) auxotrophic selection required.
2: Not added where leucine (LEU2) auxotrophic selection required.
3: Not added where MET25 promoter induction required.
4: Not added where uracil (URA3) auxotrophic selection required.
2.4: S. cerevisiae strains The wild-type S.cerevisiae strain used for all experiments was the diploid strain BY4743 (EUROSCARF; MATa/MAT his3 1/his3 1 leu2 0/leu2 0 met15 0/MET15 LYS2/lys2 0 ura3 0/ura3 0). All single gene deletants were obtained from the International Saccharomyces Gene Deletion Project (Winzeler et al, 1999). These strains were otherwise isogenic to BY4743 except the gene of interest was disrupted via insertion of the KanMX4 cassette.
2.5: Synthetic oligonucleotides Oligonucleotides for PCR were obtained from Sigma-Genosys, Invitrogen, or GeneWorks. Oligonucleotides were resuspended in TE buffer upon receipt, and used without further purification. Oligonucleotides used in this study are summarised in Table 2.3
Table 2.3: Oligonucleotide primers used in this study. Restriction enzyme recognition sites within primers are underlined, and the corresponding enzyme listed in the ‘Restriction enzyme’ column.
Name Sequence (5’ – 3’) Restriction enzyme /$), )))+)+)...)++)..).+.))+ #! /$)* .+).)).)+.++.+)).++++ ! /$)*& .+).)).)+.+))+.+)).++++ ! /$)*%>) .+).)).)+.+)..))+.++++ ! /$) * .))).)+.)+.+.++++.++++ ! /$) *& .)))..)+.+)).++++.++++ ! +8, )+.).))+)..+))+++.))) *!
33 +8* +.+)..)+.+)...++++.)..) ! $&+>*, )+)).+)..+)+.)+..)).. ! $&+>** +.)))+.)+)....))))))+)++)) ! )>, .++..)++)..+)..++++.++.. #! )>* .++.)+.+++++).+))..++... !
2.6: General lab procedures All heat-stable apparatus, media and reagents were sterilised by autoclaving for 15 minutes at 120oC and 125 kPa. Heat labile solutions were sterilised via filtration through disposable 0.22 μm sterile filters (Millipore). All waste was disposed of in accordance with the regulations of the University of New South Wales and appropriate government authorities.
2.7: General molecular biology procedures
2.7.1: Small scale plasmid DNA miniprep purification For small scale preparation of plasmid DNA for molecular biological purposes, 5 ml overnight cultures of E. coli were grown at 37oC in LB media supplemented with the appropriate antibiotic. Plasmid DNA was purified using the PureLinkTM Quick Miniprep Kit (Invitrogen) according to the manufacturer’s instructions. Purified DNA was eluted from columns in 10 mM TE buffer and used immediately, or stored at -20oC until required.
2.7.2: Large scale plasmid DNA preparation Larger quantities of plasmid DNA were prepared for transformation of 96-well plate arrays of S. cerevisiae strains using the following method. 50 mL E. coli cultures were grown overnight at 37oC in LB media supplemented with the appropriate antibiotic for selection. Cells were pelleted by centrifugation at 4000 r.p.m. for 15 min. at 4oC, and the supernatant removed. The cell pellet was resuspended in 2 mL of GTE buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris, pH 8.0) and incubated at room temperature for 5 minutes. Cells were lysed by addition of 4 mL of lysis solution (0.2 M NaOH, 1% SDS), and gently mixed by inverting the tube end over end until all cloudiness disappeared and the solution turned viscous. 3 mL of ice-cold 3 M potassium acetate solution was added, gently mixed, and the slurry incubated on ice for 5 min. The slurry was transferred to a centrifuge tube, and centrifuged at 12000 r.p.m. for 30 min at 4oC in a Sorvall SS-34 rotor. The supernatant was transferred to a new tube, 5.4 mL of isopropanol was added, incubated for 2 min at room temperature, and centrifuged again at 12000 r.p.m. for 30 min. at 4oC in a Sorvall SS-34
34 rotor. The supernatant was removed, and the isopropanol pellet washed and resuspended in 1 mL of aqueous 70% (v/v) ethanol. The suspension was centrifuged at 14000 r.p.m. for 5 min, the supernatant removed, and the pellet dried in a Savant SpeedVac for 10 min. The DNA pellet was resuspended in 500 μL of TE buffer.
2.7.3: Agarose gel electrophoresis Routine analysis of DNA was performed using agarose slab gels (1% - 2% w/v, depending on application) cast with 1 x TAE buffer and ethidium bromide (800 ng/l). Electrophoresis was performed using a horizontal submarine-type apparatus (Gibco) at a constant voltage of 80 V until the desired separation was achieved. DNA was visualised using an ultra-violet transilluminator (254 nm wavelength) or BioRad GelDoc XR system.
Where separated DNA fragments were excised from the gel and used for further reactions, ultra-pure low melting point agarose (GeneWorks) was substituted, and ultra-violet transillumination was performed at 309 nm wavelength.
2.7.4: Determination of DNA concentration DNA concentration was determined spectrophotometrically using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, USA). 2 μl of sample was analysed, and DNA considered to be acceptably pure if the A260/A280 ratio was between 1.8 and 2.0.
2.7.5: Polymerase Chain Reaction (PCR) PCR was used for amplification and mutagenesis of DNA. iProofTM (BioRad) or PhusionTM (Fermentas) proof-reading high fidelity polymerases were used for all reactions. Typical reactions contained 50 – 300 μg of template DNA, 0.2 mM of each dNTP (Roche), 0.5 μM of each primer, 1 x PCR reaction buffer (as supplied by the manufacturer) and 1 unit of polymerase per 50 μl reaction. Where the manufacturer supplied reaction buffer did not contain magnesium chloride, it was supplemented to a final concentration of 1 mM.
Reactions were performed using a DNA Engine Tetrad (MJ Research) thermal cycler, equipped with a heated lid and Peltier cooled heat blocks. Typical cycling conditions consisted of 98oC denaturation step for 1 min, followed by 30 cycles of denaturation at 98oC for 10 s, annealing at 50 – 70oC for 30 s, and extension at 72oC for 30 – 60 s. A final
35 extension step of 72oC for 10 min was included. Successful amplification was confirmed via agarose electrophoresis.
2.7.6: Restriction digestion All restriction enzymes were obtained from New England Biolabs. Restriction digests were performed under the conditions recommended by the manufacturer, using the supplied buffers and supplements. Where required, digests were terminated by heat inactivation, or by purification of DNA fragments using a Qiagen QIAquickTM PCR purification kit, according to the manufacturer’s instructions. Successful digestion of DNA was confirmed via agarose electrophoresis.
2.7.8: Ligation of DNA All DNA ligations were performed using T4 DNA ligase from New England Biolabs. Ligation was typically performed using 20 units of T4 DNA ligase in a 1x concentration of T4 ligase buffer (containing ATP) as supplied by the manufacturer. For liquid phase ligations, reactions were incubated for 1 h at room temperature, or overnight at 4oC as required before transformation into E. coli.
Ligations were also performed using DNA embedded in agarose slices excised from gels, as required. As mentioned in Section 2.7.3, ultra-pure low melting temperature agarose was used to cast gels in these situations. Gel slices containing DNA fragments of interest were melted by incubation in a 65oC water bath for 10 min. Molten aliquots of agarose containing insert and vector DNA were combined in the desired ratio, and cooled for 5 min in a 37oC water bath. T4 ligase and T4 ligase buffer were added in the appropriate amounts, and the reaction allowed to proceed overnight at room temperature. Ligated DNA was received by melting the agarose gel plug at 65oC for 10 min.
2.7.9: Creation of chemically competent E. coli Chemically competent DH-5 or XL-10 strains of E. coli were generated by treatment with rubidium chloride. An overnight starter culture of cells was grown in LB media at 37oC. A 50 ml LB culture was inoculated from the starter culture (get volume) and grown at 37oC until it reached an OD600 of 0.6. Cells were cooled on ice for 15 min, and pelleted at 4000 r.p.m. for 10 min. at 4oC. The supernatant was removed and the pellet resuspended in 30
36 ml of ice cold RFI buffer (100 mM RbCl, 50 mM MnCl2.4H2O, 30 mM potassium acetate,
10 mM CaCl2.2H2O, 15% v/v glycerol, pH 5.8) and incubated on ice for 1 h. Cells were then pelleted at 4000 r.p.m. for 10 min. at 4oC, supernatant removed, and resuspended in 5 ml of ice cold RFII buffer (10 mM MOPS, 10 mM RbCl, 15% v/v glycerol, pH 6.5) for 15 min. Aliquots of 50 μl were dispensed into pre-chilled microcentrifuge tubes and snap- frozen via dry ice/ethanol bath prior to storage at -80oC.
2.7.10: Transformation of E. coli Chemically competent E. coli cells produced as described in section 2.7.9, or commercially supplied (DH-5 , Invitrogen; XL-10 Gold, Stratagene) were used for all transformations. Cells were stored at -80oC until required.
Frozen 50 μl aliquots of E. coli were thawed on ice for 10 min. 1 – 5 μl of DNA solution was added, and cells incubated on ice for a further 30 min. Cells were heat shocked for at 42oC for 30 s, and then cooled on ice for 2 min. 200 μl of LB media was added to cells, followed by a recovery incubation at 37oC for 1 h. Cells were plated onto LB plates containing the appropriate antibiotic for selection and incubated overnight at 37oC.
2.8: Construction of plasmids A listing of all vectors and constructs used in this thesis is summarised in Table 2.5. All constructs were confirmed by restriction enzyme digestion and DNA sequencing.
Table 2.5: Vectors used in this study
Vector name Source Description
>. < %!& + $., - ; ! "#$ >.< F 5 $., - ; ! "#$ >.<4 F + $., - ; %& ! "#$ >.<" F 5 $., - ; %& ! "#$ $&+>* & ,1.7 - ; μ% %& ' ( )' ( * - *& ". & μ% %& '+ 1 G C !
37 9' 1 G <: "B F ? ; μ% %& '+ "B F 4<) ; μ% %& '+ "B < F < ; μ% %& '+ "B F + ., ? ; μ% %& '+ "B 4 F + ., 4<) ; μ% %& '+ "B " F + ., < ; μ% %& '+ &56 ., !% + $., ; μ% +&!*'9+ ( %# " @: $*." * H. + * $ "; > - ( %# ', &? )( % &9/ ": *&= H . # ? / % * ! &6 *&=< 1 F # < 1 >. <.1 ) + $., - ; ' ( >.<., I + ! "#$ >.<"., I + %& ! "#$ >.<4.1 F + $., - ; %& ' ( >. <.17 F + $., ; ' ( >.<4.17 F + $., ; %& ' ( >.<4.17 $ F + $., !$7 ; %& ' ( >.<4.17 F + $., ; %& ' ( 6 >.<4.17? F + $., ? ; %& ' ( >.<4.17< 1 F + $., < 1 ; %& ' (
1: Map of this vector displaying additional detail shown in Figure 2.1.
38 2.8.1: Construction of pUG34-GFP and pUG36-GFP The pUG collection of plasmids was obtained from MIPS (University of Dusseldorf, Germany; http://mips.gsf.de/proj/yeast/info/tools/hegemann/gfp.html). The pUG collection is intended for the construction of N- and C-terminally EGFP-tagged proteins for expression in S. cerevisiae, under the control of the moderate strength, methionine- repressible MET25 promoter.
Derivatives of pUG34 and pUG36 lacking the EGFP sequence 5’ of the multiple cloning site were created for the expression of non-EGFP tagged proteins. The XbaI-flanked EGFP coding region was removed from by digestion of pUG34 or pUG36 DNA with XbaI, followed by self-ligation of the plasmid to create pUG34-GFP and pUG36-GFP, respectively.
2.8.2: Construction of pUG23GAL1 and pUG35GAL1 Derivatives of pUG23 and pUG35 containing the stronger galactose-inducible GAL1 promoter substituted for the original MET25 promoter, were created as follows. The 485bp GAL1 promoter was PCR amplified using pESC-URA (Stratagene) plasmid DNA as a template, with forward primer ESC-URA-F (containing a SacI site) and reverse primer ESC-URA-R (containing a XbaI site). pUG35 DNA was digested with SacI/XbaI to release the MET25 promoter fragment, and after digestion with the same enzymes, the GAL1 promoter was ligated into pUG35 to produce pUG35GAL1.
2.8.3: Construction of pUG23GAL1/A 1-42 and pUG35GAL1/A 1-42 The coding sequence for the 42 amino acid amyloid beta 1-42 (A 1-42) fragment was amplified using pAS1N.A GFP (Caine et al, 2007) plasmid DNA as a template (a gift from Ian Macreadie, CSIRO) with forward primer ABETA-F (containing a BamHI site) and reverse primer ABETA-R (containing a SalI site). The purified 159bp A 1-42 PCR product was digested with BamHI and SalI, and ligated into pUG35GAL1 digested with the same enzymes to create pUG35GAL1/A 1-42 (Figure 2.1).
2.8.4: Construction of pUG35GAL1/A 1-42-EP
A mutant form of A 1-42 with codons corresponding to the C-terminal amino acids I41 and
A42 altered to E41 and P42 was created via PCR amplification from the pAS1N template with
39 the ABETA-F forward primer and the mutagenic ABETA-R-MUT reverse primer. The resulting PCR product was designated A 1-42-EP. The A 1-42-EP product was ligated into pUG35GAL1 as described in the previous paragraph to create pUG35GAL1/A 1-42- EP (Figure 2.1).
2.8.5: Construction of pUG35GAL1/A 1-40 A 3’ truncated form of A 1-42 with the last 2 codons removed was created via PCR amplification from the pAS1N.A GFP template with the ABETA-F forward primer and the mutagenic ABETA-140-R reverse primer. The resulting PCR product was designated A 1- 40. The A 1-40 product was ligated into pUG35GAL1 as above to create pUG35GAL1/A 1-40 (Figure 2.1).
40
2.8.6: Construction of pUG34-GFP/A 1-42 A native, unfused form of the A 1-42 peptide was created by PCR amplification of the A 1-42 coding region from the pAS1N.A GFP template using the ABETA-F forward primer and ABETA-R-S reverse primer (containing both a stop codon and a SalI restriction site). The resulting PCR product was digested with BamHI and SalI and ligated into pUG34-GFP digested with the same enzymes to create pUG34-GFP/A 1-42.
41 2.8.7: Construction of pUG35GAL1/wt-tau and pUG35GAL1/P301L-tau The coding sequences of the wild-type and P301L mutant forms of human tau (MAPT) were PCR amplified from the pRSV-tau and pRSV-P301L plasmid DNAs as templates, respectively (kindly donated by Jurgen Gotz, Brain and Mind Research Institute, University of Sydney). Forward primer TAU-F (containing a BamHI site) and reverse primer TAU-R (containing a SalI site) were used. The resulting PCR products were digested with BamHI/SalI and ligated into pUG35GAL1 cut with the same enzymes to yield pUG35GAL1/wt-tau and pUG35GAL1/P301L-tau, respectively.
2.9: Transformation of S. cerevisiae
2.9.1: Transformation of individual S. cerevisiae strains Starter cultures of S. cerevisiae were incubated overnight in YEPD at 30oC, and then inoculated into fresh 5 ml YEPD cultures at an OD600 of approximately 0.1. Cultures were o grown at 30 C until an OD600 of 0.5 - 0.7 was reached. Cells were pelleted, washed with 10 ml of sterile MilliQ water, followed by pelleting and washing with 1 ml of 100 mM lithium acetate. Cells were resuspended in 500 μl of 100 μ lithium acetate and divided into 50 μl aliquots. Aliquots were pelleted, supernatant removed, and 240 μl 50% (w/v) PEG-3350, 36 μl 1 M lithium acetate, 25 μl 4 mM single-stranded herring sperm DNA, 5 μl plasmid DNA and 34 μl MilliQ water added in order to each aliquot. Cell pellets were resuspended in the transformation mix by vortexing and pipetting. Cells were incubated at 30oC for 30 min, followed by heat shock at 42oC for 30 min. Cells were pelleted, transformation mix removed, and pellets resuspended in 200 μl MilliQ water prior to plating onto SC plates supplemented with an amino acid mixture appropriate for selection. Plates were incubated at 30oC for 2 - 4 d until colonies were clearly visible.
2.9.2: Transformation of 96-well plate arrays of S. cerevisiae strains Frozen 96-well plates were thawed at room temperature for approximately 25 min before being replicated (using a sterile 96-pin replicating tool) into sterile 96-well round-bottom microtitre plates (Greiner) containing 160 μl YEPD media per well. Plates were incubated at 30oC shaking at 600 r.p.m. for 2 d. Cells were pelleted, supernatant removed, and 20 μl sterile MilliQ water added per well. Cells were resuspended into the water by shaking at 900 r.p.m. for 1 min. 160 μl of transformation mix was added per well (40% w/v PEG-
42 3350, 0.1 M lithium acetate, 1 mM EDTA, 10 mM Tris-HCl pH 7.5, 20 ng/ml single stranded herring sperm DNA), supplemented with the appropriate plasmid DNA to a concentration of approximately 5 μg per well. Plates were shaken at 900 r.p.m. for 1 min to completely mix cells into the transformation mix, and then incubated at 30oC for 1 d. Cells were heat-shocked at 42oC for 15 min, pelleted, and the supernatant removed. 150 μl of SC media (minus the appropriate nutrients for selection of the appropriate plasmid-borne auxotrophic marker) was added per well, and cells incubated at 30oC shaking at 600 r.p.m. for 2 d. As a second round of selection, a 5 μl aliquot of each cells from each well was transferred to a fresh 96-well plate (containing 150 μl per well of selective SC media) and incubated for a further 24 h at 30oC with shaking at 600 r.p.m. Plates were scored for successful transformants at this stage. Transformants were stored by resuspending cell pellets in 15% (w/v) glycerol and freezing at -80oC.
2.10: Screening of 96-well plate arrays for A -GFP fluorescence and localisation 96-well plates containing yeast cells transformed with the pUG35GAL1/A 1-42 plasmid (encoding the A 1-42-GFP fusion protein; Section 2.16.3) were replicated (using a 96-pin replicating tool) into 96-well microtitre plates containing 150 μL per well of SC media (with 2% w/v galactose as a carbon source and lacking uracil for plasmid selection). Microtitre plates were grown with shaking at 900 r.p.m. at 30oC for approximately 24 h. 5 μl aliquots of cells were removed from each well and examined for the appearance of A 1- 42-GFP fluorescence using fluorescence microscopy (see Section 2.15). A minimum of several thousand cells were typically observed from each aliquot. A strain was scored as a preliminary hit if it displayed a level of GFP fluorescence deemed to be higher than the wild-type control expressing the pUG35GAL1/A 1-42 construct. Relatively lenient thresholds were applied at this stage in order to not exclude any potential hits, at the cost of an increased false-positive rate.
Any strain scored as a preliminary hit was subcultured in 5 mL of SC-ura/2% (w/v) galactose media and grown overnight before being re-examined more closely with fluorescence microscopy. At this stage, strains exhibiting a substantial level or distinctive localisation of A 1-42-GFP fluorescence were photographed (Section 2.15) and considered
43 as a confirmed hit.
2.11: Bioinformatic analysis of S. cerevisiae mutant genes Lists of mutants identified in Section 2.9 were analysed for the appearance of co-occurring functional groupings by the GENECODIS software (Carmona-Saez, et al., 2007). GENECODIS is available at the URL http://genecodis.dacya.ucm.es. Annotations for GO Biological Process, GO Molecular Function, GO Cellular Component and KEGG Pathways were selected for inclusion in the analysis. As the entire S. cerevisiae genome was not considered in the A 1-42-GFP fluorescence screen, a custom reference list, consisting of the 3680 mutants which were successfully transformed and observed was used.
Mapping of S. cerevisiae genes to human homologs was performed using the NCBI HomoloGene database, available at the URL http://www.ncbi.nlm.nih.gov/sites/entrez?db=homologene. All other analyses of S. cerevisae genes and associated annotations were performed using the Saccharomyces Genome Database and associated tools, available at the URL http://www.yeastgenome.org.
2.12: Protein extraction from S.cerevisiae
50 ml cultures of S .cerevisiae were grown to an appropriate OD600 and pelleted by centrifugation. Cell pellets were washed once with ice cold MilliQ water, pelleted, and then resuspended in 200 μl of ice cold breakage buffer (100 mM Tris, pH 8.0, 20% glycerol) supplemented with 1 x protease inhibitor cocktail (CompleteTM EDTA-free, Roche) and 10 mM EDTA. An approximately equal volume of glass beads was added to the slurry, and cells were lysed by processing in a mini-bead beater for 3 times 1 min. at 4oC, interspersed with 1 min of cooling on ice. An additional 200 μl of breakage buffer was added, and the sample clarified by centrifugation at 13 000 r.p.m. in a benchtop mini centrifuge for 10 min at 4oC. The resulting supernatant, containing total cellular soluble proteins, was collected and protein concentration determined by the Bradford dye-binding assay (see Section 2.11). Protein samples were processed for SDS-PAGE immediately, or stored at -80oC until required.
44 2.13: Determination of protein concentration Protein concentration was determined via the Bradford dye-binding assay. Bovine serum albumin (BSA) was used as a standard. Absorbances were determined using a SpectraMaxTM 400 96-well plate reader (Molecular Dynamics). In cases where a protein sample contained detergents that interfered with the Bradford assay, the BioRad DC Protein Assay Kit was used in accordance with the manufacturer’s instructions.
2.14: Polyacrylamide gel electrophoresis Protein samples were prepared for SDS-PAGE separation by addition of loading buffer to a 1x final concentration (1% SDS, 10% glycerol, 125 mM Tris pH 6.8, 0.01% bromophenol blue). Where reducing conditions were required, samples were further supplemented with 1% -mercaptoethanol. Samples were incubated in a 100oC water bath for 5 minutes and centrifuged at 13,000 r.p.m. for 5 min prior to loading onto the gel. NuPAGETM Bis-Tris polyacrylamide gradient gels with NuPAGETM MOPS running buffer in an XCell SureLockTM apparatus (Invitrogen) were used for separation.
2.15: Electroblotting of SDS-PAGE gels Proteins separated via SDS-PAGE were transferred to nitrocellulose membranes for Western analysis via electroblotting. Blotting was performed using an Invitrogen XCell IITM blotting apparatus. The transfer buffer was a 1x solution of bicine/bis-Tris Invitrogen NuPAGE Transfer Buffer, supplemented with 10% (v/v) methanol and 0.01% SDS. A transfer voltage of 30 V for 1 h was used. Satisfactory transfer of proteins to the nitrocellulose membrane was confirmed by staining of the membrane with Ponceau S (0.1% w/v Ponceau S, 5% v/v acetic acid). Membranes were destained by thorough washing with reverse osmosis water.
2.16: Western blotting and enhanced chemiluminescence (ECL) detection All incubations were carried out at room temperature with gentle agitation. The primary antibodies used in this study are summarised in Table 2.4.
Table 2.4: Primary antibodies used in this study
Antibody Supplier Antigen Antibody Species Working Name type concentration .,9,1: &+ -. J
45 ., "$ +- / J "
All antibodies were diluted to the appropriate working concentrations in 1x TBS-T buffer. Volumes of antibody solutions were chosen to ensure good coverage of the membrane surface area and to avoid drying out. Membranes were blocked in 2% (w/v) blocking agent (GE Life Sciences) in TBS-T for 1 h. Blots were incubated in primary antibody for 1 hour, and then washed 4 times for 5 min in large volumes of TBS-T. Blots were incubated with HRP-conjugated secondary antibody for 1 h, followed by another 4 washes for 5 min in large volumes of TBS-T. Blots were incubated with ECL reagents (GE Healthcare) as recommended by the manufacturer, and then exposed to high sensitivity film (HyperFilmTM, GE Life Sciences) for a period of time determined to produce an optimal image.
2.17: Fluorescence microscopy and staining All light and fluorescence microscopy was performed using an Olympus BX-60 microscope equipped with an Olympus mercury arc lamp. S. cerevisiae cells were typically observed using the 100x 1.3 NA oil immersion lens and phase contrast optics. Images were electronically captured using a cooled black and white CCD camera (Princeton Instruments), capture interface card (Roper Scientific) and IPLab software. Images were acquired at a resolution of 1300 x 1030 pixels and saved as 16-bit greyscale TIFF files. Conversion to 8-bit RGB TIFF images, false colouring and multiple image overlays were performed using the NIH ImageJ v1.38 software (http://rsb.info.nih.gov/ij). Additional operations including cropping and resizing were performing using Photoshop CS3 software (Adobe).
4’,6’-diamidino-2-phenylindole (DAPI; Sigma) was used to detect nuclear and mitochondrial DNA via fluorescence microscopy. Cells were pelleted, supernatant removed, and the pellet resuspended in water. DAPI was added to a final concentration of 45 μg/ml and cells incubated at room temperature in the dark for 20-30 min. Cells were examined directly under the fluorescence microscope using the ‘WU’ filter set.
46
LipidTOX RedTM (Invitrogen) was used to stain neutral lipids such as those present in lipid droplets. Yeast cells were stained with LipidTOX RedTM by dilution of the manufacturer- supplied 1000x dye stock to a final concentration of 1x, in an aliquot of cells harvested directly from a growing culture. The cells were vortex mixed and incubated in the dark for 20-30 min before being examined with the 'SWG' filter set.
47 3: A fluorescence-based reporter of intracellular amyloid- beta aggregation in Saccharomyces cerevisiae
3.1: Introduction and aim The successful formation of the chromophore of green fluorescent protein (GFP) when expressed as a C-terminal fusion to another protein, is proportional to the ability of the upstream protein to properly fold, and avoid the formation of insoluble aggregates or inclusion bodies (Waldo, et al., 1999). Observation of the fluorescent intensity of a GFP fusion therefore provides a direct report of protein folding and solubility. This reporter can be adapted to a range of qualitative and quantitative techniques including fluorescence microscopy, spectrofluorimetry and fluorescence activated cell sorting (FACS).
The propensity towards aggregation of the Alzheimer’s Disease amyloid beta (A ) peptide is one of the fundamental aspects of its pathobiology. This makes the peptide highly amenable to analysis by expression and observation of an A -GFP fusion. Hecht and colleagues have performed a series of experiments using such A -GFP fusions, expressed in Escherichia coli. These include the discovery of mutations in the A 1-42 peptide that reduced (Wurth, et al., 2002) or increased (Kim and Hecht, 2008) its amyloidogenic properties, the contribution of C-terminal residues to the increased amyloidogenicity of A 1-42 relative to A 1-40 (Kim and Hecht, 2006), and the identification of compounds via high-throughput assay which inhibit A 1-42 aggregation (Kim, et al., 2006). In each of these experiments, a straightforward measurement of A -GFP fluorescence could be directly related to an increase or decrease in the aggregation of the fusion protein. These findings were supported by another laboratory, which also generated a large set of A 1-42 mutants fused to GFP to investigate the role of specific amino acid side chains present in the central hydrophobic region of the peptide on its aggregation (de Groot, et al., 2006).
The major focus of A research has traditionally centred on the insoluble extracellular amyloid plaques, which are a hallmark of Alzheimer’s Disease. As discussed in Section 1.2.7, the relatively recent confirmation of bona fide intracellular pools of A peptide has resulted in a surge of interest in the role of A in this context. The development of
48 informative cellular and organismal models of intracellular A aggregation and toxicity is key to discovering the nature of its involvement in the development and progression of Alzheimer’s Disease. The yeast Saccharomyces cerevisiae is a widely used model of eukaryotic cell biology, characterised by its well defined and tractable genetics, rapid growth, and significant sequence and functional homology to higher organisms. A number of recent publications have described the use of S.cerevisiae for the study of neurodegenerative diseases, including Huntington’s Disease (Krobitsch and Lindquist, 2000; Meriin, et al., 2002; Willingham, et al., 2003), Parkinson’s Disease (Willingham, et al., 2003; Outeiro and Lindquist, 2003), and Alzheimer’s Disease (Vandebroek, et al., 2005; Bagriantsev and Liebman, 2006) and report the recapitulation of key disease-related phenotypes in this organism. These studies validate S. cerevisiae as a useful cellular model of various neurodegenerative diseases, particularly those involving protein aggregation.
Therefore, the aim of the research described in this chapter was the development of a model of intracellular A aggregation and localisation in S. cerevisiae, based on the expression of an A -GFP fusion protein. This model exploits the tendency of aggregation-prone fusion domains to inhibit the correct folding of GFP, resulting in the abolition of fluorescence. Measurement of A -GFP fluorescence consequently acts as a simple measure of the degree of A misfolding and aggregation. The development of this model in S. cerevisiae facilitates the future development of large-scale screening protocols, useful for discovering genetic and environmental factors affecting intracellular A aggregation and localisation, in a well-defined eukaryotic organism.
3.2: Results
3.2.1: An A 1-42-GFP fusion protein is not fluorescent in yeast To determine whether the N-terminal addition of A 1-42 to GFP resulted in the production of a fusion protein with diminished or abolished fluorescence in the cytosol of S. cerevisiae, a galactose-inducible expression plasmid encoding this fusion (pUG35GAL1/A 1-42; see Section 2.8.3) was constructed and transformed into wild-type cells and observed via fluorescence microscopy.
49 Trace levels of A 1-42-GFP associated fluorescence were detected in a small proportion of exponential growth phase cells (OD 0.4 or OD 1.0) (Figure 3.1). This fluorescence was typically barely distinguishable from background signal levels. Where discernable, the fluorescence appeared to be distributed throughout the cell, although it was not possible to assign a localisation with any degree of confidence at this level of signal. The majority of cells displayed no fluorescence. No additional A 1-42-GFP fluorescence was detected in cells in stationary phase (data not shown).
In contrast, control cells expressing GFP alone (transformed with the pUG35GAL1 plasmid; Section 2.8.2) under the same conditions exhibited a strong green fluorescent signal. GFP was evenly distributed throughout the cell, with the notable exception of compartments presumed to be vacuoles (Figure 3.2). This localisation is consistent with previous observations of GFP expressed in wild-type S. cerevisae (Niedenthal et al, 1996). Fluorescence of the unfused GFP was stable, remaining clearly observable in a high proportion of cells even 24 hours into stationary phase.
50
Figure 3.1: Galactose-induced expression of the A 1-42-GFP fusion protein (from the pUG35GAL1/A 1-42 plasmid) in exponential phase wild-type BY4743 cells. Left panel: Cells observed with phase contrast light microscopy. Right panel: Same cells observed using fluorescence microscopy. Fusion of A 1-42 to GFP resulted in minimal fluorescence
51 3.2.2: A less amyloidogenic mutant form of A 1-42-GFP is fluorescent To examine whether the minimal green fluorescence observed in cells expressing A 1-42- GFP was caused by aggregation of the fusion protein, and not merely by inefficient expression or rapid turnover, a known non-aggregating form of A 1-42-GFP was studied. A series of mutations at residues 41 and 42 of the A 1-42 polypeptide have been identified which lead to enhanced fluorescence (and hence reduced amyloidogenicity) when expressed in E. coli (Kim and Hecht, 2006). Substitution of isoleucine 41 to glutamic acid, and of alanine 42 to proline resulted in the greatest reduction of amyloidogenicity, and therefore these two mutations were introduced into a variant of the A 1-42-GFP expression construct, to create pUG35GAL1/A 1-42-EP (see Section 2.8.4 for details).
Wild-type BY4743 cells expressing the A 1-42-EP-GFP mutant fusion, observed during exponential growth phase (OD ~ 1.0), displayed a distinct green fluorescent signal which was distributed throughout the cell (figure 3.3). The overall appearance of these cells was comparable to cells expressing GFP alone (figure 3.2). This result is consistent with the dramatically enhanced fluorescence of the same I41E / A42P mutant form of A 1-42-GFP reported by Kim and Hecht (2006), and demonstrates that when expressed in S. cerevisiae, the GFP portion of a less amyloidogenic A 1-42-GFP fusion is able to efficiently form the chromophore, thus producing robust green fluorescence.
In some instances, a higher concentration of green fluorescent signal could be observed in a region of the cell presumed to be the nucleus. This may indicate that the more soluble A 1-42-EP-GFP mutant is preferentially transported to the nuclear membrane, or into the nucleus itself. As unfused GFP is distributed throughout the cell (with the exception of the vacuole) , this result may indicate that a specific property of the A 1-42-EP portion of the fusion is responsible for its nuclear localisation.
52
Figure 3.2: Galactose-induced expression of unfused GFP (from the pUG35GAL1 vector) in exponential phase wild-type BY4743 cells. Left panels: Cells observed using phase contrast light microscopy. Right panels: Same cells observed using fluorescence microscopy.
53
Figure 3.3: Expression of the mutated Ab1-42-EP-GFP fusion protein (from the pUG35GAL1/Ab- EP plasmid) in exponential wild-type cells. Left panels: Cells observed with phase contrast light microscopy. Right panels: Same cells observed using fluorescence microscopy.
54
3.2.3: An A 1-40-GFP fusion protein exhibits fluorescence As a further confirmation that the S. cerevisiae-based system can allow differentiation between forms of A -GFP with differing tendencies to aggregate, a C-terminally truncated form of the A peptide lacking residues 41 and 42, A 1-40, was produced as a GFP fusion (pUG35GAL1/A 1-40; see Section 2.8.5). A 1-40 is among the peptide species produced by the variable processing of the amyloid precursor protein by gamma-secretase in the amyloidogenic pathway (Section 1.2.4). A 1-40 has a lower propensity to form aggregates compared to the more hydrophobic A 1-42 (Jarrett, et al., 1993). Therefore, an A 1-40- GFP fusion represents a pathologically relevant positive control, which could be expected to exhibit more fluorescence compared to A 1-42-GFP.
When expressed in BY4743 wild type cells and observed during exponential growth (OD ~ 1.0), the A 1-40-GFP fusion exhibited an obvious green fluorescence, in excess of that observed for the A 1-42-GFP fusion (Figure 3.4). However, the intensity of this fluorescence was lower overall than that observed for GFP alone, or for the A 1-42-EP- GFP fusion. This indicates that a level of aggregation of the A 1-40-GFP fusion is occurring that is somewhere between A 1-42-GFP, and A 1-42-EP-GFP or GFP alone. This finding is consistent with studies demonstrating that while the A 1-40 peptide is far less amyloidogenic than A 1-42, it is still capable of self-aggregation (Jarrett, et al., 1993), and that this pathologically relevant property is able to be faithfully reproduced in the S. cerevisiae GFP fusion model.
As was found for GFP and A 1-42-EP-GFP, the A 1-40-GFP fusion was distributed throughout the cell, with the exception of the vacuole (Figure 3.2). Additionally, small intensely fluorescent punctate patches could be observed in certain cells. Although these punctate structures did not appear localised within a particular organelle, they were frequently observed to be in close association with a cell structure that is most likely the nucleus. This is reminiscent of the observation that the soluble mutant A 1-42-EP-GFP was sometimes found concentrated in the nucleus, and may indicate that the A peptide has
55 an affinity for the nucleus or nuclear membrane in yeast. As the A 1-42-GFP is not fluorescent, it is not possible to conclude from these data if the nuclear localisation of A 1- 40-GFP and A 1-42-EP-GFP is due to their increased solubility, or if this localisation is a common property of all A isoforms.
3.2.4: Fluorescence levels of GFP, A 1-40-GFP and A 1-42-GFP are distinguishable via flow cytometry An attractive feature of the GFP fusion model for studying protein aggregation is that it may be adapted for use with quantitative methods such as flow cytometry. The results described above demonstrate that forms of the A peptide with known differences in their amyloidogenicity can be distinguished from each other via fluorescence microscopy. In order to complement these qualitative findings with quantitative data, flow cytometry was performed.
Samples of BY4743 wild-type cells expressing A 1-42-GFP, A 1-40-GFP or GFP alone were collected during exponential phase (OD ~ 1.0) and analysed via flow cytometry. 3000 cells were analysed for each construct. As demonstrated in Figure 3.5, the flow cytometer was capable of clearly distinguishing the fluorescent signal emitted from each of the 3 different cell populations. The FL1 fluorescence channel histogram confirms the ranking of fluorescence that was observed via fluorescnce microscopy, with the A 1-42-GFP, A 1- 40-GFP and GFP-only expressing populations exhibiting minimal, moderate and large degrees of fluorescence, respectively. An extended shoulder is observed for the A 1-40- GFP histogram relative to that for A 1-42-GFP , likely reflecting heterogeneity in the amount of diffuse cytosolic fluorescence, and the number and size of fluorescence puncta. This result, while preliminary, supports the use of technologies such as flow cytometry for the rapid and quantitative analysis of experiments performed using the yeast GFP-fusion assay system.
56
Figure 3.4: Expression of the truncated A 1-40-GFP fusion protein (from the pUG35GAL1/A 1-40 plasmid) in exponential phase wild-type cells. Left panel: Cells observed using phase contrast light microscopy. Right panel: Same cells observed using fluorescence microscopy.
57
Figure 3.5: Differences in A fluorescence can be clearly distinguished using flow cytometry. Overlaid flow cytometry histograms of FL1-channel GFP fluorescence (x-axis) and cell count (y- axis) of wild-type cells expressing unfused GFP (red), A 1-40-GFP (blue) and A 1-42-GFP (green). 3000 cells were counted for each type.
3.2.5: Lack of A 1-42-GFP fluorescence is not due to lack of translation, cleavage, or destruction of the fusion protein A potential explanation for the diminished levels of A 1-42-GFP fluorescence compared to unfused GFP is that the fusion protein is inefficiently translated, or rapidly degraded. To test for this possibility, protein extracts were prepared from cells expressing A 1-42-GFP, A 1-40-GFP and unfused GFP, and western blots were performed using anti-GFP and anti- A antibodies.
In the blot probed with anti-GFP antibody (Figure 3.6a), the unfused EGFP control (lane 1) was strongly detected at an approximate molecular mass of ~ 28 kDa, which is in good agreement with the theoretical molecular mass of EGFP (26.8 kDa). Distinct bands were detected in lanes 2 (A 1-40-GFP) and 3 (A 1-42-GFP) at an apparent molecular mass of approximately 36 kDa. The predicted masses of A 1-40-GFP and A 1-42-GFP are 31.7
58 kDa and 31.9 kDa respectively. The minor discrepancy between the predicted and observed molecular masses of the A -GFP fusion proteins may be attributable to the adoption of a non-linear conformation of the aggregation-prone A region, even in the presence of SDS. The persistence of an anomalously migrating, aggregation-related conformation may also explain the slightly larger apparent molecular mass observed for the A 1-40-GFP fusion versus the A 1-42-GFP fusion, even though A 1-40-GFP is 184 Da lighter. An alternative explanation for the larger than expected apparent molecular masses is the attachment of unknown post-translational modifications to the fusion proteins.
After discounting non-specifically detected proteins which were present in all three lanes, there were no other major bands observed that would indicate the presence of partial breakdown products of GFP, A 1-40-GFP or A 1-42-GFP. Importantly, there was no evidence for the cleavage of A 1-40 to release free GFP, which could then correctly fold and be responsible for the significantly higher amounts of fluorescence observed in cells expressing the A 1-40-GFP fusion protein.
These results are confirmed in the blot probed with anti-A antibodies (Figure 3.6b). The observed molecular masses for the detected bands are identically to those estimated from the anti-GFP probed blot, and only a single major band is detected for both A 1-40-GFP and A 1-42-GFP samples. In this case, electrophoresis was performed so as to ensure that the small (approximately 4 kDa) A fragment would not run out of the bottom of the gel if it produced via its cleavage from the fusion protein. No small molecular weight bands are able to be detected which would indicate the presence of free A peptide.
The relative intensity of the band corresponding to A 1-40-GFP was greater than that of A 1-42-GFP when blots were probed with either anti-GFP or anti-A antibodies. This may reflect an increased solubility of the A 1-40-GFP fusion protein versus A 1-42-GFP, which resulted in a higher recovery in the soluble protein extract loaded onto the gels. To further test this hypothesis, protein extracts should be prepared from the pellet remaining after soluble protein extraction (using a combination of detergents and potentially chaotropes such as urea to facilitate recovery of less insoluble A 1-42-GFP) and western 59 blotting performed. It would be expected that an increased signal level for A 1-42-GFP would be observed from this less soluble pellet fraction relative to that from the soluble fraction.
Taken together, the results demonstrate that the A -GFP fusion proteins are correctly translated in S. cerevisiae, and are not rapidly processed to unfused GFP, or completely degraded.
60
Figure 3.6: The A 1-42-GFP fusion protein is detectable via western blotting in non-fluorescent cells. a: Load-controlled soluble cell extracts from wild-type cells expressing the indicated protein were separated on SDS-PAGE gels, electroblotted, and probed with anti-GFP antibodies. Both A 1-40-GFP and A 1-42-GFP fusion proteins are detected at the correct molecular mass. b: Same extracts probed with anti-A antibody (6E10). The GFP control lane is clear, and the positions of the A -GFP fusion proteins correspond exactly to those in the anti-GFP probed blot.
61 3.2.6: Expression of A 1-40-GFP or A 1-42-GFP is not detrimental to the growth of yeast Intracellular A has been demonstrated to have toxic effects in a number of experimental systems, including human neuronal cell culture (Zhang, et al., 2002), transgenic mice (Billings, et al., 2005; Oakley, et al., 2006; Van Broeck, et al., 2008) and Drosophila brain (Crowther, et al., 2005), and is proposed to play a key role in the early development of AD (reviewed in Laferla, et al., 2007). While the results presented above show that physiologically relevant aspects of A amyloidogenesis are recapitulated in Saccharomyces, the application of this model would be broader if intracellular A -GFP also caused toxicity. Therefore, growth and spot test experiments were performed to determine if the expression of A -GFP fusion proteins had a deleterious effect on growth.
No differences in growth rate were observed between BY4743 wild-type cells expressing A 1-42-GFP or A 1-40-GFP when compared to cells expressing unfused GFP (data not shown). This indicates that toxicity related to expression of A -GFP is so minor that there is no effect on growth, or that any toxic effects can be dealt with adequately by the innate defense mechanisms of the cell. This result is largely consistent with other studies of A - GFP behaviour in S. cerevisiae, where expression of C-terminal and N-terminal fusions of GFP to A 1-42 resulted in reductions in growth of 5% and 4%, respectively (Caine et al, 2007). Similarly, substitution of the prion-forming N-terminal domain of Sup35p with A 142 resulted in no detectable change in growth when expressed in S. cerevisiae (von der Haar et al, 2007). The detection of only intact full length A 142-GFP and A 1-40- GFP protein via western blotting (Section 3.2.5) discounts rapid degradation of the fusions as an explanation for the lack of effect on growth rate.
Metals such as copper, zinc and aluminium have been implicated in the progression of Alzheimer’s Disease and in promoting A related accumulation and/or toxicity (reviewed in (Barnham and Bush, 2008; Rodella, et al., 2008). To determine if copper or aluminium was capable of unmasking a toxic effect of the A 1-42-GFP fusion protein in S. cerevisiae, spot growth tests were performed on media containing a range of concentrations of either copper sulphate or aluminium chloride. As for cells grown in unsupplemented media, no
62 difference in growth rate was detected between cells expressing A 1-42-GFP, A 1-40- GFP, or unfused GFP alone in the presence of copper or aluminium ions (data not shown).
Although A -GFP toxicity would represent a useful expansion in the scope of the S. cerevisiae model, a side benefit is that genetic and environmental effects on the aggregation of A -GFP may be studied in isolation from any confounding toxic effects the fusion protein may have.
3.2.7: Expression of unfused A 1-42 does not inhibit growth, and the peptide can not be detected As noted in Section 3.2.6, A -GFP fusions expressed in S. cerevisiae display relevant aggregation-related behaviours, but do not result in significant toxicity. This lack of toxicity is in contrast to the notable deleterious effects of intracellular A 1-42 in various other model systems and organisms. While it is possible that the innate stress response mechanisms of Saccharomyces provide the cell with adequate protection against any A - GFP related toxicity, another possibility is that the fusion of GFP to A eliminates any potential toxic effects that the peptide may cause when expressed in Saccharomyces. To test for this possibility, an unfused version of the A 1-42 peptide was created (Section 2.8.6).
Expression of unfused A 1-42 in wild-type BY4743 cells did not result in a reduction of growth, compared to an empty-vector control (data not shown). While A 1-42-GFP does not appear to be rapidly degraded (Section 3.2.5), such a fate was still possible for the unfused A 1-42 peptide. Western blots were performed on extracts from cells expressing the unfused A 1-42 peptide, using anti-A antibodies (Section 2.16). As shown in Figure 3.6, no A 1-42 could be detected using this approach. It is unlikely that the lack of detectable peptide is due to a vector or expression-related issue, since the same plasmid vector was used for expression of A 1-42-GFP, which is clearly identifiable via western blotting. A more likely explanation for the lack of toxicity of unfused A 1-42 in S.cerevisiae is that the peptide is rapidly degraded. Although fusions of A 1-42 to GFP (Caine et al, 2007), maltose-binding protein (Macreadie et al, 2007) and the Sup35 MRF
63 domain (Bagriantsev and Liebman, 2006; von der Haar, 2007) are all able to be detected via western blotting when expressed in S. cerevisiae, specific efforts to detect expression of the unfused peptide in yeast have failed (Caine et al, 2007; von der Haar et al 2007). Taking these results with those presented in this thesis, it appears that when expressed in the cytoplasm of S. cerevisae, unfused native A 1-42 peptide is rapidly destroyed, but N- and C-terminal fusions of the peptide to a range of other sequences protect it from degradation.
3.2.8: Exposure to guanidine hydrochloride results in appearance of A 1-42- GFP fluorescence Guanidine hydrochloride (GuHCl) is capable of “curing” the natively occurring prions of S. cerevisiae by inhibiting the activity of the ATPase subunit of Hsp104p, a heat shock protein essential for the propagation of prions in yeast (Chernoff, et al., 1995). Hsp104p contributes to prion inheritance by shearing and fragmenting aggregates of prion proteins, allowing their successful partitioning and propagation into daughter cells. (Kryndushkin, et al., 2003; Shorter and Lindquist, 2004). The aggregation-prone nature of prions is similar in many respects to that of A . Heat shock proteins (HSPs) are involved in many aspects of A pathobiology, including direct interaction with the peptide (Fonte, et al., 2002), modulation of A 1-40 and A 1-42 production and secretion (Yang, et al., 1998), and providing protection from the toxic effects of A in vivo (Yan, et al., 1997; Fonte, et al., 2002; Fonte, et al., 2008). Therefore, wild-type cells expressing A -GFP were treated with guanidinium hydrochloride to examine if the Hsp104p-mediated prion curing effects of this chaotropic agent were also capable of affecting the fluorescence, and hence folding, of the A 1-42-GFP fusion.
When grown in the presence of 10 mM GuHCl, a concentration known to result in prion curing (Tuite, et al., 1981) and inhibition of Hsp104p (Jung and Masison, 2001), wild-type cells expressing A 142-GFP exhibited multiple highly fluorescent punctate patches, together with a degree of diffuse cytoplasmic fluorescence (Figure 3.7). The puncta were mostly distributed throughout the cytoplasm, with some clustering occurring in and around the nucleus, which is a similar localisation to that observed in cells expressing the A 140- GFP fusion. The appearance of such fluorescence indicates that GuHCl is capable of
64 influencing the amyloidogenic properties of the A 142-GFP, rendering the fusion protein less likely to form fluorescence-inhibiting aggregates.
The pronounced effect of an externally added chemical agent on the fluorescence of the A 142-GFP fusion protein indicates that this system may also be suitable for the screening of drug and chemical libraries for compounds with anti-amyloidogenic properties.
3.2.9: The anti-amyloidogenic effect of guanidine hydrochloride acts through a Hsp104p-independent mechanism As noted above, the ability of GuHCl to prevent prion propagation is mediated through its inhibitory effect on the ATPase activity of Hsp104p. To determine if the dramatic increase in A 142-GFP fluorescence in cells treated with 10 mM GuHCl was also due to inhibition of Hsp104p activity, A 142-GFP was expressed in a homozygous diploid mutant strain with the HSP104 gene deleted. hsp104 mutant cells expressing A 142- GFP exhibited no GFP fluorescence, as per wild-type cells (data not shown). This result indicates that the absence of Hsp104p is insufficient to lead to the appearance of A 142- GFP fluorescence, and that the effects of GuHCl are mediated through a pathway which is independent of the inhibition of Hsp104p.
65 Figure 3.7: Exposure of wild-type cells expressing the A 1-42-GFP fusion protein to 10mM guanidine hydrochloride (GuHCl). Left panels: Cells observed using phase contrast light microscopy. Right panels: Cells observed using fluorescence microscopy. GuHCl exposure results in a dramatic increase in A 1-42-fluorescence. Examples of diffuse cytoplasmic (a), nuclear (b) and small punctate (c) and large punctate (d) fluorescence are all present, as indicated by arrows.
66 3.3: Discussion This chapter describes the construction and characterization of a fluorescence-based reporter system for studying the aggregation of the amyloid beta peptide in an intracellular, eukaryotic environment. The system represents an extension of the original E. coli based A -GFP fusion reported by Hecht and coworkers (Wurth et al, 2002; Kim and Hecht, 2006; Kim et al, 2006; Kim and Hecht, 2008) and de Groot, et al., 2006.
An initial concern was that the strong inhibition of chromophore formation caused by fusion of the A 1-42 peptide to GFP when expressed in E. coli by Hecht and colleagues may not be repeatable in S. cerevisiae. Compared to prokaryotes, eukaryotic cells have a greater capacity to co-translationally fold multidomain proteins in their cytosol (Netzer and Hartl, 1997). This capacity helps avoid the formation of the misfolded, insoluble inclusion bodies which are frequently observed when exogenous multidomain proteins are expressed in prokaryotes. S. cerevisiae is capable of efficiently folding a number of GFP fusion proteins which are poorly folded in E. coli (Chang et al, 2005), and thus A 1-42-GFP fusions which are non-fluorescent when expressed in E. coli, might have been able to efficiently fold and fluoresce in a eukaryote such as S. cerevisiae. However, the marked reduction of A 1-42-GFP fluorescence in S. cerevisiae (Section 3.2.1) proves that the disruptive effect of the A 1-42 sequence upon chromophore formation in GFP is not dependent on expression in a prokaryotic system. Additionally, the moderately and strongly enhanced fluorescence of A 1-40-GFP (Section 3.2.3) and A 1-42-EP-GFP (Section 3.2.2) respectively, demonstrate that a eukaryotic system is also capable of faithfully representing the subtleties between different forms of A as seen in a prokaryotic system (Hecht and Kim, 2005).
A preliminary study of the ability of the S. cerevisiae A -GFP fusion system to quantitatively distinguish differing forms of the A peptide was performed using flow cytometry (Section 3.2.4). Qualitative differences, as determined by visual assessment of fluorescence micrographs, were clearly reflected in flow cytometry fluorescence histograms. It is not yet known what effect the appearance of a non-uniform pattern of fluorescence (eg: the range of small to large puncta observed in wild-type A 1-42-GFP
67 cells treated with 10mM GuHCl; Figure 3.7) would have on the accuracy of flow cytometry based quantitation, and this requires further investigation. It may be possible using flow cytometry analysis to identify separate populations of cells containing different types of A -GFP fluorescence, such as those appearing after GuHCl treatment. This would provide a rapid method of quantitating not only absolute levels of fluorescence, but also sub- populations of cells containing specific types of A -GFP localisation and aggregation. Coupled to a cell-sorting function, cells displaying A -GFP fluorescence patterns of interest may be separated for further downstream analyses, such as protein extraction and western blotting. In addition to flow cytometry, a pilot experiment was also conducted to test the ability of a 96-well microtitre plate spectrofluorimeter to distinguish cultures expressing differently fluorescent A -GFP constructs. While only a preliminary result, it was shown that it was possible to differentiate between different A -GFP constructs using a spectrofluorimeter. Further experiments aimed at refining this approach should be conducted, as the ability to utilise a 96-well fluorescence assay would be highly advantageous for applications such as screening drug and chemical libraries for compounds which alter A -GFP aggregation.
3.3.1: Comparison with other Saccharomyces cerevisiae-based models of A aggregation As the research described in this thesis was in progress, a number of S. cerevisiae-based models of A aggregation were published by other groups. Together, these publications provide much corroborating and complementary evidence to that presented here. The section below interprets many of the main results in this chapter in light of these additional publications.
3.3.1.1: An A -GFP fusion S. cerevisiae model Of particular relevance is the report of the construction, expression and characterization of an A 1-42-GFP fusion in wild-type S. cerevisiae by Caine et al (2007). The work of Caine et al sought to develop a yeast model of A biology which is similar in some aspects to that detailed in this thesis. However, in contrast to the abolition of fluorescence caused by the fusion of A 1-42 to GFP reported here, expression of the A 1-42-GFP construct developed
68 by Caine et al (pAS1N.A GFP) resulted in the appearance of intensely fluorescent puncta, distributed throughout the cell and in association with lipid droplets.
A potential explanation for this discrepancy is the presence of a relatively lengthy linker region (22 amino acids) between the C-terminus of A 1-42 and the N-terminus of GFP in the pAS1N.A GFP construct. The linker length of all A -GFP constructs used in this study is only 4 amino acids. The strikingly different levels of A 1-42-GFP fluorescence resulting from expression of the two different constructs indicates that linker length may influence the ability of A to inhibit the correct formation of the GFP chromophore. The differing properties of the short and long linker versions of A 1-42-GFP can act as complementary tools for examining A behaviour within the cell. Since the short-linker form described in this thesis is not normally able to be visualised via fluorescence microscopy, it is difficult to determine its intracellular localisation. The use of amyloid-specific stains such as thioflavin T was investigated in this study, but due to the confounding influence of the yeast cell wall, these experiments were unsuccessful. The localisation described by Caine et al (2007) of their long-linker fusion may provide clues as to the actual localisation of the non-fluorescent, short-linker A 1-42-GFP.
As in this study, expression of the A 1-42-GFP fusion by Caine et al (2007) resulted in a mild reduction in growth yield (4% - 5%). Interestingly, it was also reported that expression of A 1-42-GFP resulted in a three-fold increase in the activation of the heat shock response, as determined by measurement of -galactosidase activity in a strain co- transformed with an A 1-42-GFP expression and HSE- -galactosidase reporter constructs. A minimal reduction in growth, despite a strongly induced stress response, adds weight to the hypothesis that while an A 1-42-GFP fusion may be inherently toxic, wild-type S.cerevisiae is capable of mounting an effective defense by way of its innate stress response systems (Section 3.2.6). If this hypothesis is correct, transformation of a panel of strains lacking genes involved in stress responses (particularly the heat shock response) and screening for reductions in growth (or induction of lethality) could identify which elements of the stress response are responsible for overcoming A 1-42-GFP toxicity. As there is significant homology between yeast and human genes involved in responses to stresses 69 such as heat shock, oxidative stress and the unfolded protein response, and deficiencies in these pathways have been implicated in the progression of Alzheimer’s Disease (reviewed respectively in Wilhelmus, et al., 2007, Sayre, et al., 2008 and Yoshida, 2007), results obtained from such a screen would be very informative.
3.3.1.2: A -Sup35p fusion S. cerevisiae models The S. cerevisiae genome encodes at least three natively occurring, non-disease associated prions ([URE3], [PSI+] and [PIN+]). As such, yeast has come under considerable experimental scrutiny from prion researchers, and a detailed understanding of prion behaviour has been developed in this organism (reviewed in Perrett and Jones, 2008). The understanding of one prion-forming protein in particular, Sup35p, has been exploited to develop an assay of A misfolding and aggregation in S. cerevisiae.
Sup35p is essential for growth in S. cerevisiae and functions as a translational termination factor, suppressing read-through of stop and nonsense codons. Sup35p is capable of forming an infectious, insoluble prion conformation, which results in conversion from a normal [psi-] state, to a phenotype of reduced translational termination efficiency known as [PSI+] (reviewed in von der Haar and Tuite, 2007). The amino acid sequence of Sup35p may be divided into three distinct functional regions. The N-terminal 123 amino acids are necessary and sufficient for Sup35p to attain the insoluble, aggregated [PSI+] prion state (Ter-Avanesyan, et al., 1994). The C-terminal region of Sup35p is entirely responsible for the essential function of translation termination. The intermediate domain of Sup35p contains a stretch of highly charged residues, and its precise function remains unclear. The N-terminal prion domain is not required for the function of the C-terminal region, and is may be completely deleted, or substituted with other sequences.
While the conversion of Sup35p to the insoluble [PSI+] prion state abolishes its translational termination function, [PSI+] results in only subtle effects on growth under normal conditions. A convenient method for assaying [PSI+] formation has been developed, by coupling the [PSI+]-mediated decrease in translational termination efficiency, to increased read-through of a premature stop codon in a mutant ade1 allele. The ade1 allele causes adenine auxotrophy (Ade-), and the accumulation of a red coloured
70 intermediate of the adenine biosynthetic pathway, leading to the growth of pink/red colonies. Read-through of the premature stop codon of ade1-14 results in the production of a functional full-length Ade1p protein, loss of adenine auxotrophy (Ade+), and the disappearance of the red coloured intermediate. Thus, [PSI+] leads to the growth of white, Ade+ colonies which are easily detected by eye.
3.3.1.3: Aggregation of A 1-42/Sup35p compared to A 1-42-GFP As the N-terminal prion-forming domain of Sup35p may be replaced with other amino acid sequences, it is possible to substitute aggregation-prone sequences, such as A , and exploit the convenient red/white [PSI+] assay system for their study. Bagriantsev and Liebman (2006) and von der Haar et al (2007) have independently reported the creation of N- terminally A 1-42 substituted Sup35p constructs and their expression in S. cerevisiae. Importantly, expression of A -substituted Sup35p in ade1 reporter strains by both Bagriantsev and Liebman (2006) and von der Haar et al (2007) resulted in the loss of adenine auxotrophy and appearance of white colonies, which indicates that the A 1- 42/Sup35p chimera is able to aggregate and achieve a [PSI+]-like prion form. This is in agreement the lack of A 1-42-GFP fluorescence reported in this thesis, and provides corroborating evidence for the aggregation of A 1-42 fusions in S. cerevisiae, via an independent assay system.
In addition, both groups examined the ability of the A 1-42/Sup35p system to distinguish A isoforms known to display altered aggregation propensities. Bagriantsev and Liebman (2006) introduced known aggregation-inhibiting mutations into the A 1-42 portion of their construct, resulting in the production of pink Ade+ colonies when expressed. It was also noted that the colour of these colonies was slightly less red than those expressing a control non-A 1-42 tagged Sup35 construct demonstrating that although these mutations were not identical to those present in the A 1-42-EP-GFP mutant described in Section 3.2.2, they provide further independent corroborating evidence that expression of known non- aggregating mutants of A in S. cerevisiae results in the expected behaviour.
71 3.3.1.4: Aggregation of A 1-40/Sup35p and A 1-40-GFP fusions The less aggregation-prone, C-terminally truncated A 1-40 was also examined by von der Haar et al. Unlike the pink [psi-] colonies which resulted from the expression of A 1-42 point mutants, only white [PSI+] colonies were observed, indicating that the A 1- 40/Sup35p fusion was still forming aggregates. This aggregation was confirmed by a sedimentation assay, which showed levels of A 1-40/Sup35p aggregation were indistinguishable from those of A 1-42/Sup35p. This contrasts with the appearance of moderate levels of diffuse and punctate fluorescence in cells expressing an A 1-40-GFP fusion (Section 3.2.3), indicative of reduced levels of aggregation as compared to non- fluorescent A 1-42-GFP. Von der Haar et al (2007) hypothesized that A 1-40/Sup35p aggregation may be due to its point-localised intracellular concentration exceeding the required threshold for efficient aggregation of A 1-40. If this is correct, the hypothesized localised regions of high A 1-40 concentration may correspond to the fluorescent puncta reported in Section 3.2.3. In this scenario, the A 1-40-GFP puncta are fluorescent despite presumably containing aggregated A 1-40-GFP, because the aggregation occurs relatively slowly, allowing sufficient time for the GFP chromophore to correctly form. The lower concentration pool of unaggregated fluorescent A 1-40-GFP would correspond to the diffuse fluorescence observed throughout the cell, and localised regions of higher A 1-40- GFP concentration, high enough to encourage aggregation, to the fluorescent puncta.
3.3.1.5: Guanidine hydrochloride, Hsp104 and A 1-42 Bagriantsev and Liebman (2006) and von der Haar et al (2007) also examined the effect of the prion-curing agent guanidine hydrochloride (GuHCl) on the aggregation of their A 1- 42/Sup35p fusions, which are directly comparable to the experiments detailed in Section 3.2.8. GuHCl exerts its prion curing effect by inhibiting the ATPase activity of Hsp104, a chaperone required for maintenance and inheritance of the [PSI+] prion (Kryndushkin, et al., 2003). von der Haar et al (2007) reported that exposure to GuHCl produced no detectable change in colony colour of A 1-42/Sup35p expressing cells, and therefore concluded that GuHCl had no effect on A 1-42/Sup35p aggregation. In contrast, Bagriantsev and Liebman (2006) reported that exposure to GuHCl in their system resulted in a marked increase in the appearance of high-n oligomeric forms of A 1-42/Sup35p, as
72 determined by SDS agarose electrophoresis and western blotting. It should be noted that the authors did not detail the effect of GuHCl treatment, as determined by the red/white colony assay. The lack of effect observed by von der Haar et al (2007) may simply reflect an inherent limitation of the red/white colony system to detect GuHCl-mediated changes. In this study, exposure to GuHCl resulted in the appearance of substantial, heterogenous A 1-42-GFP fluorescence (Section 3.2.8 and Figure 3.7). This fluorescence took the form of variably sized puncta distributed throughout the cell, diffuse cytoplasmic fluorescence, and some fluorescence localised to an unknown organelle, presumably the nucleus. The punctate structures may correspond to the high-n oligomers detected by Bagriantsev and Liebman (2006), although due to the size of the larger puncta, it is likely that aggregation of A 1-42-GFP continues beyond the high-n oligomeric state. As the appearance of fluorescence is necessarily indicative of correct GFP chromophore formation, GuHCl may reduce the rate of A 1-42-GFP aggregation sufficiently to allow time for productive folding of the GFP chromophore.
To confirm this hypothesis, puncta would need to be purified and examined by a similar agarose electrophoresis technique to that utilised by Bagriantsev and Liebman (2006) to determine their oligomeric state. It is worth noting that free puncta have been observed via fluorescent microscopy in the semi-clarified supernatant of glass-bead lysed A 1-42-GFP expressing cells (data not shown). It may be possible to design a relatively simple purification strategy for the puncta, based upon cell lysis and differential centrifugation.
The appearance of substantial amounts of diffuse cytoplasmic A 1-42-GFP fluorescence after GuHCl treatment is consistent with the hypothesis that GuHCl may reduce the rate of A 1-42 oligomer formation. A reduced rate of oligomer or aggregate formation would lead to the accumulation of a pool of correctly folded, fluorescent monomeric A 1-42-GFP. It is interesting to note that the putative nuclear localisation of the A 1-42-GFP in GuHCl treated cells is identical to that observed in cells expressing the less aggregation-prone A 1-40-GFP construct. Nuclear localisation may be a characteristic of A peptides with reduced rates of aggregation. Again, SDS-agarose electrophoretic analysis of purified nuclei from cells exhibiting the A -GFP nuclear localisation (ie: those expressing A 1-40-
73 GFP or A 1-42-GFP and treated with GuHCl) should be performed to resolve this uncertainty.
To determine if the marked effects of GuHCl on A 1-42 aggregation were due to inhibition of Hsp104p, Bagriantsev and Liebman (2006) also examined the oligomerisation state of A 1-42/Sup35p in a hsp104 mutant, via SDS-agarose electrophoresis. A lack of Hsp104p lead to a increase in the proportion of monomeric versus oligomeric A 1- 42/Sup35p. Exposure of the hsp104 mutant to GuHCl resulted in the reappearance of a larger proportion of high-n A 1-42/Sup35p oligomers. Taken together, these results indicate that the pro-oligomerisation effects of GuHCl are mediated via a Hsp104p- independent mechanism. These results are consistent the those presented in Section 3.2.9, as A 1-42-GFP fluorescence was not observed in hsp104 cells, thus providing an additional independent confirmation.
3.3.1.6: Toxicity of A 1-42/Sup35p and A 1-42-GFP fusions No detrimental effect on growth was detected after the expression of the A 1-42/Sup35p fusion protein by von der Haar et al (2007). Exposure to 1 mM copper sulphate, 3 mM hydrogen peroxide, or 1 mM diamide (an agent known to cause glutathione depletion and oxidative stress in S. cerevisiae) also failed to inhibit the growth of A 1-40/Sup35p expressing cells. Taken together with the results presented in Section 3.2.6 and the observations of Caine et al (2007), these data provide compelling evidence that the cytosolic expression of an A 1-42 fusion with either GFP or Sup35p, is not sufficiently toxic to cause major reductions in growth. Interestingly, while Caine et al (2007) demonstrated that expression of A 1-42-GFP caused a threefold induction of the heat shock response, von der Haal et al (2007) did not detect any generation of reactive oxygen species after expression of A 1-42/Sup35p, using the ROS-sensitive dye dihydroethidium (DHE). The cellular effects of cytosolic A 1-42 expression in S. cerevisiae may be limited to the induction of the heat shock response, without the associated production of ROS that is observed in other cellular models of A 1-42 toxicity.
74 3.3.1.7: Comparative strengths and weaknesses of A 1-42/Sup35 and A 1- 42-GFP fusion systems The red/white colony [PSI+] assay represents an elegant method of detecting A 1-42 aggregation. Because the output of the assay is a simple change in colony colour, an obvious advantage of this system is the ease with which changes in A aggregation may be detected. Colony colour change can be readily adapted to various high-throughput screening approaches. An example may be the transformation of an A -Sup35p reporter strain with a gene overexpression library, and screening for genes which alter the aggregation of A . In this case, commercially available automated colony picking robots could be configured to recognise white colonies, automatically selecting them for further analysis and identification of the overexpressed gene. While a screen of this type is also possible with an A -GFP system, it would require the use of a fluorescence activated cell sorter to identify and separate cells exhibiting increased A -GFP fluorescence. Separated cells would then be re-plated prior to picking of single colonies for identification of the overexpressed gene leading to A -GFP fluorescence.
In comparison to the A 1-42/Sup35p system, the A -GFP fluorescence approach offers a two distinct advantages. First, as non-aggregated A -GFP can be visualised with fluorescent microscopy, it is possible to determine its intracellular location without further chemical or immunological staining. Secondly, because the A 1-42/Sup35p reporter is reliant on the presence of an ade1 allele with a premature stop codon, this limits the range of host strains which are compatible with the assay. For example, the EUROSCARF yeast genome knockout collection has been created in a BY4743 strain background, and this strain does not carry the ade1 allele. This genetic incompatibility precludes the use of the A 1-42/Sup35p system with a gene knockout screen experiment. As the A -GFP fusion system is not dependent on a specific genetic background, it does not suffer from this limitation; the transformation of an A 1-42-GFP construct into the entire yeast genome knockout collection to screen for genetic factors affecting A aggregation is described in the following chapter.
75 4: A genome-wide screen for modifiers of A 1-42 aggregation and localisation
4.1: Introduction and aims The previous chapter discussed the development and characterization of a fluorescence- based reporter of intracellular A misfolding and aggregation in S. cerevisiae. This reporter is based upon expression of an A 1-42-GFP fusion protein. The presence of the rapidly aggregating A 1-42 domain inhibits the correct formation of the GFP chromophore, rendering the fusion protein non-fluorescent. Conditions which affect the aggregation of the A 1-42 domain, such as deletion or mutation of residues 41 and 42, may result in the appearance of GFP fluorescence (Sections 3.2.2 and 3.2.3). Similarly, specific changes in the intracellular environment brought about by external factors, such as guanidine hydrochloride exposure, also result in A 1-42-GFP fluorescence (Section 3.2.8). The appearance of fluorescence, and its detection and quantitation by techniques such as flow cytometry and fluorescence microscopy, provides a simple and powerful method for the assay of intracellular A aggregation. An additional benefit of this system is that the intracellular localisation of a fluorescent A 1-42-GFP fusion may also be conveniently determined.
The amyloid cascade hypothesis posits that accumulation of A is a fundamental event in the pathogenesis of AD, which triggers a wide-ranging cascade of cellular and physiological events, eventuating in massive neuronal death, loss of synaptic connections and deterioration of mental and cognitive function. Extracellular A in various forms has been the focal point of this hypothesis, first as the conspicuous insoluble amyloid plaques, and then later as the soluble oligomeric species. The detection of intracellular pools of A , and the observation that these pools may appear prior to the development of other pathological signs of AD has resulted in renewed interest in the relatively poorly characterised intracellular A species. However, knowledge of the cellular mechanisms which are involved in the production, trafficking, localisation, oligomerisation, degradation and toxicity of intracellular A remains incomplete. Given the key role that is emerging for intracellular A peptides in the development of AD, elucidation of these mechanisms is
76 critical. Alterations in the intracellular metabolism of A peptide have been proposed as a potential therapeutic method for treating AD. Transgenic animal and cell models are utilised as relevant models in which to examine A pathobiology. However, despite recent advances in RNAi gene-silencing techniques, and the ongoing development of resources such as the mouse genome knockout collection, these models remain less than ideal for the large scale, high-throughput screening of genetic factors affecting perturbations of cellular physiology, such as that occurring in AD.
As discussed in Section 1.4, S. cerevisiae provides a powerful and flexible platform for such genome-wide screening experiments. Such screens have been performed using humanized yeast models of Huntington's and Parkinson's Disease, expressing a mutant huntingtin protein and -synuclein, respectively (Willingham, 2003, p03516; Outeiro and Lindquist, 2003). The identification of gene knockouts which enhanced the toxicity of either of these disease associated genes resulted in the development of an integrated model for the processes which were involved in the cellular response. Many of these genes had human orthologues, providing a launching point for more targeted studies in mammalian models.
Therefore, the aim of this study was to identify and characterise changes in cellular physiology which affect the intracellular aggregation and/or localization of A 1-42. This was achieved via transformation of the mutants in the S. cerevisiae homozygous diploid deletion mutant collection with an A 1-42-GFP expression plasmid, and inspection of each strain with fluorescence microscropy. As A 1-42-GFP in wild-type yeast is misfolded and thus non-fluorescent, changes in the behaviour of A 1-42 brought about by deletion of a specific gene will be indicated by the appearance of GFP fluorescence.
Acknowledgement: The transformation, screening and characterization of the yeast gene deletion collection described in this chapter was performed in conjunction with Suresh Nair, an honours student in the School of Biotechnology and Biomolecular Sciences, UNSW.
77 4.2: Results
4.2.1: Transformation of the Saccharomyces genome knockout collection Strains from the Saccharomyces genome knockout collection were transformed with the A 1-42-GFP reporter plasmid (pUG35GAL1/A 1-42) as described in Section 2.8.2. This transformation protocol dealt with strains on a plate-by-plate basis, maintaining each individual mutant in a distinct microtitre well throughout the entire procedure. This is in contrast to other mass-transformation approaches which have been described, which are based on "one-pot" transformations of pooled populations of strains (Willingham, et al., 2003). While these approaches greatly simplify the task of transformation, they necessarily result in mixed pools of transformants, and rely on the assay procedure to provide a method of rapidly determining the identity of mutants of interest. In this case, microscopic examination of each strain to detect changes in A 1-42-GFP localisation was required, discounting a mixed pool transformation strategy. An advantage of the plate-by-plate method is that there is now a library of transformed strains which are easily retrieved for future experiments, without requiring a selection or identification step.
In total, 3680 strains were successfully transformed, representing approximately 80% of the entire gene knockout collection and 56.2% of all 6540 predicted ORFs in the S. cerevisiae genome. Varying levels of transformation efficiency were noted from plate to plate, which may reflect the broad range of fitnesses and tolerance to the transformation protocol in the mutant collection. The relatively crude plasmid DNA preparation protocol which was used to generate the large quantities of DNA required (Section 2.7.2) did not appear to negatively affect the success of subsequent transformations relative to plasmid DNA prepared using a commercially available midi-prep kit (data not shown). This plasmid preparation protocol offered the advantages of low-cost, simplicity to perform, and reasonable yield of DNA.
4.2.2: Screening for A 1-42-GFP fluorescence and localisation After successful transformation with the pUG35GAL1/A 1-42 vector, A 1-42-GFP expression was induced by replicating the transformants into media containing 2% (w/v) galactose (as described in Section 2.9). After growth for 14 - 18 hours, strains were individually examined via fluorescence microscopy for the appearance of A 1-42-GFP 78 fluorescence. Strains exhibiting fluorescence were re-cultured and re-examined to confirm the original observation. Of 3680 strains which were successfully transformed with the pUG35GAL1/A 1-42 vector, 94 strains were confirmed as exhibiting A 1-42-GFP fluorescence (2.6% of transformed strains).
The identity of these 94 single gene knockout strains, together with a description of A 1- 42-GFP localisation is summarised in Table 6.1 (see 6: Appendix). These strains are grouped into broad functional categories, based upon annotations retrieved from the current version of the Saccharomyces Genome Database. Functional categories that are enriched in the screen compared to the S. cerevisiae genome as a whole may indicate cellular processes that are related to A 1-42-GFP fluorescence, and hence A misfolding and aggregation. In order to identify enriched categories, GENECODIS software was used (Section 2.11; Carmona-Saez, et al., 2007), based on a customised reference gene set consisting of the 3680 knockout strains actually screened rather than the entire S. cerevisiae genome. This custom reference gene set was used to avoid inadvertent introduction of any pre-existing functional biases in the subset of genes which were actually screened.
Using combined GO Biological Process, GO Molecular Function, GO Cellular Component and KEGG annotations, GENECODIS identified a number of gene groups in the query set that were significantly (p <= 0.05) enriched versus the reference gene set (Table 4.1).
Table 4.1: Functional categories and cellular components determined to be significantly (p <= 0.05) over-represented by GENECODIS software. Results are grouped by the ontology or annotation set considered by GENECODIS in the analysis.
Description Annotation ID p-value Genes
GO Biological Process
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+ 9)+: 0 6 "( + ,'+#+ 0 1 2%!
+ 9)+:L 0 0 @ 6 <( < + 1 2%! * - ( 9+' ( :
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80 The majority of gene classes determined by GENECODIS to be most significantly over- represented (using GO Biological Process, GO Molecular Function and KEGG as annotation sources) were related to the tricarboxylic acid (TCA) cycle, or to regulation of gene expression (via transcriptional activation and repression, or chromatin remodelling). A cluster of genes annotated with the GO Biological Process term of "protein complex assembly" was also deemed to be over-represented, although not at the same level of significance (p = 0.02). Upon further analysis, all three members of this class were determined to play roles in the assembly of components of the mitochondrial electron transport chain (ETC) (COX16 required for assembly of cytochrome c oxidase (Carlson, et al., 2003); ATP11 required for assembly of and subunits into F1F0 ATP synthase (Ackerman, 2002); CBP3 required for assembly of ubiquinol cytochrome-c reductase (Shi, et al., 2001). The identification of these ETC mutants together with those involved in the TCA cycle combine to indicate a role for mitochondrial dysfunction in the appearance of A 1-42-GFP fluorescence (discussed further in Sections 4.2.3 and 4.3.3).
A variety of A 1-42-GFP appearances and localisations were observed across the 94 mutants which exhibited fluorescence (Table 6.1). The most common appearance of A 1- 42-GFP (74 of 94 mutants) was in one or more punctate dots, ranging in size from small circular "pin-prick" points to large irregularly shaped aggregates (a similar range of puncta were observed in wild-type cells expressing A 1-42-GFP and treated with GuHCl; Figure 3.7). As discussed in Section 3.3.1.4, these punctate structures may represent aggregates or accumulations of A 1-42-GFP which formed after the correct folding of the GFP chromophore. Alternatively, they may be due to localisation of A 1-42-GFP into a specific cellular compartment or organelle. Further experimental work is required to understand the nature of these structures. A large number of mutants also exhibited a diffuse cytosolic A 1-42-GFP fluorescence (44 of 94 mutants) similar to that observed in wild-type cells expressing the truncated A 1-40-GFP (Figure 3.4) or the mutant A 1-42-EP-GFP (Figure 3.3) forms of the fusion protein. In some cases the appearance of puncta and diffuse cytosolic fluorescence occurred simultaneously (26 of 94 mutants), possibly indicating an equilibrium between freely distributed A 1-42-GFP in the cytosol and aggregated or
81 accumulated A 1-42-GFP in puncta. Fluorescence and localisation details specific to each mutant are discussed in more detail below.
4.2.3: Tricarboxylic acid cycle and oxidative phosphorylation GENECODIS analysis of the knockout mutants displaying A 1-42-GFP fluorescence revealed that genes involved in the tricarboxylic acid (TCA) cycle were the most significantly over-represented functional group as assessed both by GO Biological Process and KEGG annotations. Despite these mutants all being present in a closely linked biochemical pathway, a range of A 1-42-GFP localisations were observed, including diffuse cytoplasmic fluorescence (kgd2 ), nuclear (fum1 ) and multiple small and large puncta (idp1 ). This diversity in A 1-42-GFP localisation may reflect differences in cellular biochemistry between the various TCA cycle mutants. Potential differences due to TCA cycle mutants are illustrated in Figure 4.1, where genes identified in the A 1-42-GFP fluorescence screen are shaded in grey.
The significant over-representation of mutants involved in processes such as the TCA cycle, oxidative phosphorylation, mitochondrial protein translation and protein import indicates that normal mitochondrial function is closely linked to the folding and aggregation of the A 1-42-GFP fusion in yeast. Indeed, 37.2% of mutants identified in the screen (35 mutants from 94; Table 6.1) are annotated in the Saccharomyces Genome Database as "exhibiting a growth defect on a non-fermentable carbon source", which is often used as an indicator of disrupted mitochondrial respiratory function. The incidence of this annotation is significantly enriched in the mutants identified in the screen, versus the set of all mutants screened (465 from 3774 mutants, or 12.3%; two-tailed P < 1 x 10-4; Fisher's exact test).
82
Figure 4.1: The tricarboxylic acid (TCA) cycle, with mutants leading to the appearance of A 1-42- GFP fluorescence highlighted in grey.
A specific example of A 1-42-GFP fluorescence and localisation in a TCA cycle mutant (fum1 ) is illustrated in Figure 4.2. In the fum1 mutant, A 1-42-GFP appeared both in a diffuse cytosolically distributed form, and as small intensely fluorescent puncta. In certain cells, A 1-42-GFP fluorescence was also concentrated in an organelle-like sphere; DAPI staining and image overlay revealed this to be the nucleus. A 1-42-GFP puncta were in some cases arranged around the nucleus, but could also be observed in other unrelated regions of the cell, making a specific perinuclear localisation unlikely. DAPI staining also reveals the location of mitochondrial DNA. Despite the respiratory defect caused by the
83 fum1 mutation, there did not appear to be any specific co-localisation of A 1-42-GFP fluorescence and mitochondrial DNA DAPI staining.
AB
C D
Figure 4.2: A 1-42-GFP fluorescence and nuclear co-localisation in fum1 fumarase mutant cells. A: light. B: A 1-42-GFP fluorescence. C: DAPI fluorescence. D: merge.
4.2.4: Chromatin remodelling and transcriptional regulation After the TCA cycle, the largest functional groups identified as being over-represented in the screen were those related to chromatin remodelling and transcriptional regulation. A 1- 42-GFP fluorescence and localisation were heterogenous within this functional group, comprising various combinations of small and large puncta, in singular or multiple numbers, and diffuse cytosolic fluorescence. This heterogeneity may reflect the diversity of cellular processes which may be affected by mutations in key upstream regulators of gene expression. The possible roles of these mutations and their effect on A 1-42-GFP expression and folding are discussed further in Section 4.3.2.
84 4.2.5: Regulation of phospholipid metabolism: ino2 , ino4 and scs2 Although not identified by GENECODIS as being statistically over-represented in A 1-42- GFP fluorescence positive mutants, a cluster of genes with functions related to the regulation of inositol and lipid metabolism was identified. These included ino2 (YDR123C), ino4 (YOL108C), and scs2 (YER120W). Cells lacking INO2 displayed distinctive patterns of small intensely fluorescent puncta, which were frequently arranged in semicircular or crescent shapes (Figure 4.3 B). The path traced by these puncta were suggestive of the outline of a spherical organelle, which is probably the nucleus. In certain cells, a minor amount of A 1-42-GFP fluorescence could be detected inside these organelles. The overall appearance of A 1-42-GFP fluorescence in ino2 was similar to that observed in cho2 and opi3 mutants (see below, Section 4.2.6). In ino4 mutants, A 1-42-GFP fluorescence was more intense in the spherical nucleus-like localisation, with small puncta occasionally decorating its circumference (Figure 4.3 D). ino4 mutants also displayed diffuse cytoplasmic A 1-42-GFP fluorescence, and larger irregularly-shaped aggregates. scs2 mutants exhibited a single, large punctate patch of A 1-42-GFP fluorescence. Unlike the ino2 and ino4 mutants, there did not appear to be any particular association of this A 1-42-GFP fluorescence with a presumed organelle of any type (data not shown). To eliminate the possibility that the fluorescent puncta observed in ino2 and ino4 mutants were due to an effect mediated via GFP, rather than the A 1-42 portion of the molecule, unfused GFP was expressed in both mutants. As illustrated in Figure 4.4, unfused GFP fluorescence was detected throughout the cell in a similar manner to wild-type cells (Figure 3.2), confirming that the characteristic appearance of puncta of A 1-42-GFP in ino2 and ino4 was not due to the GFP portion of the fusion.
85 A
B
C
D
Figure 4.3: Representative light and fluorescence micrographs of ino2 (A, B) and ino4 (C, D) mutants expressing A 1-42-GFP.
86 ino2 ino4
light
fluorescence
Figure 4.4: Representative light and fluorescence micrographs of ino2 and ino4 mutants expressing unfused GFP. GFP fluorescence is distributed throughout the cell in both mutants similarly to wild-type cells, with no evidence of the fluorescent puncta illustrated in Figure 4.3
INO2 and INO4 are annotated in the GO Biological Process classification as being involved in transcriptional regulation (Table 6.1, Table 4.1). Unlike many other genes identified in this screen which were co-assigned to this classification, INO2 and INO4 are not known to be involved in the SAGA-ADA / SWR regulation of the GAL1 gene. Rather, Ino2p and Ino4p form a heterodimer which is involved in the transcriptional de-repression of a large family of genes related to phospholipid metabolism and synthesis (Ambroziak and Henry, 1994), in response to inositol depletion in the external media. Both the Ino2p and Ino4 subunits are required for full activity of the regulatory complex; neither is capable of forming a functional homodimer independently. Therefore, the detection of A 1-42-GFP fluorescence in both ino2 and ino4 mutants cross-confirms the result, and is consistent with the known interdependent nature of the Ino2p/Ino4p complex.
87 It is interesting to note that while ino2 and ino4 exhibited overlapping A 1-42-GFP localisations (localisation of small puncta arranged around the circumference of the nucleus), differences were also apparent (eg: ino4 displaying higher levels of cytosolic fluorescence and occasionally larger irregular fluorescent puncta). It is not known if these variances reflect phenotypic differences between ino2 and ino4 , or are attributable to subtle differences in experimental technique, such as the precise point in growth phase when cells were observed.
SCS2 is also involved in a regulatory system related to inositol levels in the external media. Scs2p is present in the ER lumen, and physically interacts with the Opi1p transcriptional repressor, acting to retain Opi1p there in the absence of extracellular inositol (Loewen, et al., 2003; Brickner and Walter, 2004). Therefore, Scs2p and Ino2p/Ino4p play complementary roles in the positive regulation of a number of genes, with Scs2p retaining the Opi1p repressor from the nucleus, and Ino2p/Ino4p directly activating transcription of these same genes. Deletions in any of INO2, INO4 or SCS2 could therefore be expected to result in similar phenotypes, and this is reflected in the observation of A 1-42-GFP fluorescence in all three mutants.
4.2.6: Phosphatidylcholine synthesis: cho2 and opi3 The fluorescence and distinctive localisation of A 1-42-GFP in the ino2 , ino4 and scs2 mutants suggested that other strains possessing mutations in phospholipid synthesis or metabolism may also exhibit similar A 1-42-GFP fluorescence. Mutant cho2 (YGR157W) and opi3 (YJR073C) strains displayed A 1-42-GFP fluorescence and a distinctive grouping of small puncta into ring or crescent-shaped localisation (Figures 4.5 and 4.6, respectively). To eliminate the possibility that this localisation was due to the GFP domain of A 1-42-GFP, cho2 and opi3 mutants were transformed with an unfused GFP expression vector (pUG35GAL1) and examined with fluorescence microscopy. GFP fluorescence was distributed throughout the cell as in the wild-type strain, confirming that the localisation of A 1-42-GFP was due to the A domain of the fusion protein.
88 light fluorescence
cho2 A 1-42-GFP
cho2 GFP only
Figure 4.5: Light and fluorescence micrographs of cho2 cells expressing GFP-tagged A 1-42- GFP, or GFP alone as a control.
89 light fluorescence
opi3 A 1-42-GFP
opi3 GFP only
Figure 4.6: Light and fluorescence micrographs of opi3 cells expressing GFP-tagged A 1-42-GFP peptide, or GFP alone as a control.
90 B
Figure 4.7: A 1-42-GFP fluorescence and perinuclear localisation in cho2 and opi3 mutant cells. A: Light. B: DAPI fluorescence. C: A 1-42-GFP fluorescence. D: Merge. The 'N' label highlights the nucleus in the cho2 mutant cell, which is weakly stained in this example.
91 The overall ring or crescent-shaped arrangement of A 1-42-GFP in cho2 and opi3 mutants was suggestive of distribution around a large, centrally positioned organelle, potentially the nucleus. To test for this possibility, cho2 and opi3 cells expressing A 1- 42-GFP were stained with DAPI to visualise the nucleus and mitochondria. Merging of DAPI and GFP channels revealed that the fluorescent A 1-42-GFP fusion protein was indeed present in a perinuclear arrangement, closely tracing the outline of DAPI staining (Figure 4.7). No localisation with DAPI-stained mitochondrial DNA was detected.
CHO2 and OPI3 encode enzymes with phosphatidylethanolamine methyltransferase (EC 2.1.1.17) and phospholipid methyltransferase (EC 2.1.1.16) activities, respectively. Cho2p and Opi3p are involved in the synthesis of phosphatidylcholine (PC), a major component of eukaryotic cellular membranes, via the trimethylation of phosphatidylethanolamine (PE). In humans, the enzyme phosphatidylethanolamine N-methyltransferase (encoded by the gene PEMT) is homologous to Cho2p and Opi3p, and catalyses all three methylation reactions involved in the conversion of PE to PC. The overall process is summarised in Figure 4.8. Cho2p catalyses the methylation of PE, with S-adenosylmethionine (SAM) acting as the methyl donor, to produce phosphatidylmonomethylethanolamine (PMME). Opi3p catalyses the subsequent two methylations, yielding phosphatidyldimethylethanolamine (PDME) and finally PC. Deletion of either the CHO2 or OPI3 genes abolishes the ability of the cell to produce PC via the trimethylation pathway, and results in significant reductions in the proportion of PC in cellular membranes (Summers, et al., 1988; McGraw and Henry, 1989), as well as widespread alterations in lipid and cellular homeostasis. An alternative route of PC synthesis is available via the Kennedy salvage pathway. As depicted in Figure 4.8, the Kennedy salvage pathway is dependent on the availability of precursor molecules such as monomethylethanolamine (MMEA), dimethylethanolamine (DMEA) or choline. Therefore, supplementation of the media with these precursors may circumvent cho2 or opi3 mutations, restoring the PC content of cellular membranes to near wild-type levels (Summers, et al., 1988; McGraw and Henry, 1989).
92
Figure 4.8: Simplified schematic representation of phosphatidylcholine synthetic pathways in Saccharomyces cerevisiae. Adapted from Carman, et al., 2007.
As the synthetic growth media used in this experiment did not contain any Kennedy pathway substrates, it is likely that the membrane composition of the cho2 and opi3 mutants was PC depleted. To test if the A 1-42-GFP fluorescence and punctate / perinuclear localisation observed in cho2 and opi3 mutants was related to the depletion of PC from cellular membranes or a downstream effect, cho2 and opi3 mutants expressing A 1-42-GFP were cultured in media supplemented with MMEA, DMEA or choline chloride, all at 1 mM concentration. Supplementation with 1 mM ethanolamine (EA), which is not converted to a methylated PE derivative by the Kennedy pathway was included as a control.
As expected, supplementation with EA had no effect on the fluorescence or localisation of A 1-42-GFP in opi3 mutants (Figure 4.9). MMEA was found to be toxic to opi3 cells, severely inhibiting their growth, and so these cultures were not examined. Media
93 supplementation with 1 mM DMEA or choline chloride resulted in the total disappearance of the fluorescent A 1-42-GFP perinuclear puncta, reverting to a wild-type appearance. Addition of any compound to opi3 cells expressing unfused GFP had no effect on its localisation. This result suggested that the appearance of A 1-42-GFP fluorescence in opi3 mutants (and potentially cho2 ) is dependent on inability of the cell to produce PC or PDME via the trimethylation pathway.
When PC synthesis is prevented, S. cerevisiae is capable of adaptation by altering the phospholipid content of its membranes. In the membranes of cells which are deprived of PC, the proportion of PE is greatly increased to compensate. In mutant strains such as opi3 , which retain the ability to synthesise partially methylated PE derivatives (eg: PMME), these normally rare species may also accumulate in response to PC depletion (McGraw and Henry, 1989). Therefore, it was possible that the appearance of A 1-42-GFP fluorescence was due to an increase in the proportion of PE or a partially methylated derivative, rather than a decrease in PC content. As discussed previously in this section, yeast are capable of uptake of precursors from the media, and incorporating them into phospholipid via the Kennedy salvage pathway. When a precursor molecule is abundant in the media, the membranes of a cell may become enriched in the phospholipid species directly derived from that precursor. Thus, to determine if enrichment of PE was responsible, wild-type cells expressing A 1-42-GFP were grown in media supplemented with 1 mM EA and examined for fluorescence. However, no A 1-42-GFP fluorescence was observed in wild-type cells after supplementation (data not shown). This suggests that the fluorescence appearing in cho2 and opi3 cells is due to an effect related to depletion of PC, rather than accumulation of PE.
94
95 Fine examination of the perinuclear crescents of A 1-42-GFP puncta in cho2 and opi3 cells revealed that in some cases, these puncta were distributed around a series of even smaller circular structures, giving the overall impression of a "ring of rings" around the nucleus (Figures 4.5 and 4.6). Cellular structures known to match this description are lipid droplets. Lipid droplets are composed of a neutral lipid core, containing species such as triglycerides and steryl esters, surrounded by a protein-rich phospholipid monolayer. Lipid droplets are frequently observed in a perinuclear distribution in S. cerevisiae.
To determine if A 1-42-GFP was co-localised with lipid droplets in cho2 and opi3 mutants, strains expressing A 1-42-GFP were co-transformed with a lipid droplet resident protein, Erg6p, which was tagged with the red fluorescent marker protein mDsRed (Binns, et al., 2006) (pERG6mDsRed, a kind gift of the Joel Goodman laboratory. See Table 2.5 for details). mDsRed has an emission spectrum which does not overlap with that of GFP, making it suitable for colocalisation studies. Fluorescence microscopy revealed that in both cho2 and opi3 mutants, A 1-42-GFP was immediately adjacent to the Erg6p-mDsRed signal, indicating a close association between A 1-42-GFP and the phospholipid monolayer of lipid droplets (Figure 4.10).
96 97
Lipid droplet biogenesis is believed to occur by budding off from the outer ER membrane (reviewed in Thiele and Spandl, 2008). It is therefore possible that some lipid droplet proteins which are residents of the membrane monolayer may originate from the ER membrane, and may also be detected there. A small amount of fluorescent signal from Erg6-mDsRed appeared in regions of the cell which did not correspond to the known morphology of lipid droplets, and this probably reflects Erg6p localised to the ER. To confirm that A 1-42-GFP / Erg6p-mDsRed localisation was occurring exclusively in lipid droplets, opi3 cells expressing A 1-42-GFP where stained with LipidTOX RedTM (LTR) and examined via fluorescence microscopy. LTR is a lipophilic dye which displays an intense red fluorescence when in the presence of neutral lipids. In addition, the emission spectra of LTR does not overlap significantly with that of GFP, making it an excellent tool for the determination of GFP-fusion / lipid droplet colocalisation sudies. Merging of the A 1-42-GFP and LTR fluorescence channels revealed that A 1-42-GFP was again very closely arranged around the circumference of lipid droplets (Figure 4.11). The LTR signal appeared only in morphologies corresponding with lipid droplets, discounting the possibility that A 1-42-GFP / lipid droplet localisation being confounded by non-specific staining of other cellular compartments by LTR.
98
99 4.2.7: icy2 Cells lacking ICY2 (YPL250C) exhibited A 1-42-GFP fluorescence, with the fusion protein being localised to a small number of large, intensely fluorescent inclusions, the majority of which were localised at the periphery of the cell. In some cases, faint diffuse fluorescence was also observed from a region of the cell suggestive of the nucleus (Figure 4.12-1B). To determine if either the intense or diffuse fluorescence was localised to the nucleus, icy2 cells were stained with DAPI. DAPI staining revealed that the fainter, diffuse fluorescence was present in the nucleus. There was no evidence of nuclear colocalisation for the more intensely fluorescent A 1-42-GFP signal (Figure 4.12-1C and Figure 4.12-1D).
As A 1-42-GFP was found to be closely associated with lipid droplets in cho2 and opi3 mutants, icy2 mutants were stained with LTR to test for this possibility. LTR staining revealed a normal perinuclear distribution of lipid droplets around the nucleus (co-stained with DAPI). A 1-42-GFP fluorescence was not associated with lipid droplets at all (Figure 4.12-2A – 2D)
As described above, in icy2 cells, A 1-42-GFP was frequently observed as a single punctate point at the cellular periphery. This particular localisation was suggestive of polarisation, potentially to a budding pole of the cell. To determine if A 1-42-GFP fluorescence was polarised, cells were stained with calcofluor white, which binds to chitinous bud scars on the cell wall. No consistent localisation of A 1-42-GFP with calcofluor white stain bud scars was observed (Figure 4.12-3A – 3D). When observed in relation to stained bud scars, A 1-42-GFP appeared randomly distributed around the celluar periphery, indicating that at least with respect to the budding poles, there was no polarisation of A 1-42-GFP.
The precise intracellular location of the intensely fluorescent A 1-42-GFP puncta in icy2 remains unresolved at this time. There is a relative paucity of experimental data relating to the function of the ICY2 gene, complicating further efforts to characterize the effects of its deletion on A 1-42-GFP fluorescence. Most data on ICY2 has come from large scale
100 genome-wide studies. Such studies have revealed that Icy2p is a potential substrate of Cdk1 (Ubersax, et al., 2003) suggesting a possible role in the progression of the cell cycle, and that ICY2 mRNA is mobilised to polysomes during the diauxic shift (Kuhn, et al., 2001) but little is known beyond this. Given the striking appearance of the large, intensely fluorescent A 1-42-GFP puncta in icy2 mutants, the wild-type function of ICY2 and the effects of its deletion on cellular physiology are worthwhile pursuing further.
101
4.2.8: slg1 and osmolarity sensing slg1 (YOR008C; also known as WSC1) mutant cells exhibited A 1-42-GFP fluorescence (Figure 4.13). The fusion protein appeared in a number of cellular locations, including the nucleus (4.13 a) distributed throughout the cytosol (4.13 b), and in large, distinct punctate structures arranged around the periphery of the cell (4.13 c-f). In some cells, a number of smaller puncta could also be observed distributed throughout the cell (4.13 h-i). SLG1, together with MID2, encode transmembrane proteins with overlapping roles in the sensing and transduction of cell wall stresses (via the PKC1-MPK1 cell integrity pathway). Such cell wall stresses arise under conditions such as cell wall remodelling due to growth or treatment with -factor, and after hypotonic shock. Although SLG1 and MID2 do not have significant sequence homology, they share a common domain architecture. This architecture is also similar to the mammalian integrin family, which are involved in linking the extracellular matrix to the internal cytoskeleton.
In order to determine if the appearance of A 1-42-GFP fluorescence in slg1 mutants was related to a defect in tolerance of low osmotic conditions, mutants were grown in the presence of 1M sorbitol and observed via fluorescence microscopy. No changes in A 1- 42-GFP fluorescence or localisation were observed in slg1 cells when grown in osmotically balanced media, versus standard media (data not shown). Therefore a slg1 - dependent defect in hypo-osmolarity sensing was not responsible for the appearance of A 1-42-GFP fluorescence. It is also interesting to note that the pbs2 mutant was identified in the screen as displaying A 1-42-GFP fluorescence. The intracellular localisation of A 1- 42-GFP in this mutant was also noted to be similar to that of slg1 (data not shown). Pbs2p is a MAP kinase kinase, and is also involved in the transduction of osmotic stress signals. Taken as a whole, these observations suggest an involvement of elements of the osmotic stress signalling pathway, or their downstream targets, in the modulation of A 1- 42-GFP folding and fluorescence, although this modulation may not strictly be linked to hypo-osmotic stress.
102 As noted above, SLG1 and MID2 play partially overlapping roles in the maintenance of cellular integrity. Therefore it would be interesting to examine the behaviour of A 1-42- GFP in a mid2 mutant. While mid2 is present in the yeast gene knockout collection, it was not transformed and screened in this study. This should be attempted in the future and may reveal further insight into the interaction of A 1-42-GFP and the cell wall stress sensing pathway.
light
c
f a i h g fluorescence b d e
Figure 4.13: Representative light and fluorescence micrographs of A 1-42-GFP fluorescence and localisation in slg1 mutant cells. a: nuclear b: diffuse cytosolic c - f: peripheral puncta h - i: other puncta. These patterns of A 1-42-GFP fluorescence were not abolished when slg1 cells were grown in the presence of 1M sorbitol.
4.2.9: Dubious ORFs The authenticity of a number of ORFs predicted to exist in the S. cerevisiae genome is considered "dubious". This consideration may arise for a number of reasons, including lack of corroborating experimental evidence, lack of sequence homology with closely related species, and refinements in bioinformatic techniques which render previous genomic annotations obsolete. Currently 815 S. cerevisiae ORFs are classified as dubious, representing 12.3% of the all ORFs in the genome. Because many dubious ORFs overlap 103 the coding regions of authentic ORFs, the Saccharomyces gene deletion colletion includes a number of strains corresponding to dubious ORF deletions, which may also contain partial disruptions of neighbouring authentic ORFs.
The A 1-42-GFP fluorescence screen identified seven strains with dubious ORFs deleted (Table 6.1). In four of these mutants, the dubious ORF region is known to partially overlap a neighbouring gene. Descriptions and functions of these neighbouring ORFs are summarised in Table 4.2 below:
Table 4.2: Identity and descriptions of authentic ORFs known to overlap dubious ORFs identified in the screen.
Dubious A 1-42-GFP Overlapping Overlapping Overlapping gene ORF Name fluorescent Gene Name ORF Name description morphology
8* 4+ + 8* C % ? *4 ? ; 8$*K+ # 8$* C ! $* - ? ,,) ' -- ( -< + = 81 C % &# 8* + + -53 & -- ; #%1 #%* 5 ; - 5 8* < C 1 #* 8* <+ % G +( (
104
In the case of YER119A-C, further analysis of this dubious ORF confirmed the identification of a neighbouring authentic ORF mutant in the screen. The scs2 mutant was identified in the screen as exhibiting A 1-42-GFP fluorescence, in the form of a single punctate structure (Table 6.1). YER119A-C overlaps the chromosomal coordinates of SCS2 by 103 base pairs, and the corresponding deletion mutant also exhibits the same single punctate fluorescence. This may provide an independent confirmation of the effect of the scs2 mutation on A 1-42-GFP fluorescence.
The remaining ORFs listed in Table 4.2 all overlap and disrupt authentic ORFs which are involved in functional processes previously identified in the screen. The most notable example is of YDR230W, which partially overlaps the COX20 gene. Cox20p is involved in the processing of Cox2p, prior to its assembly into the cytochrome c oxidase complex. Although cox20 was not explicitly identified in the screen, a cluster of genes involved in closely related ETC complex assembly processes were (Table 4.1; ATP11, CBP3 and COX16), and were determined to be significantly over-represented. YDR230W also displayed the same diffuse cytosolic A 1-42-GFP fluorescence as cox16 , which is involved in the same cytochrome c oxidase complex assembly. Thus, scrutiny of the genomic neighbours of dubious ORFs may provide corroborative evidence for the role of certain processes in the appearance of A 1-42-GFP fluorescence.
4.3: Discussion Intraneuronal pools of the A peptide play an important, yet unclear role in the pathogenesis of AD. The assembly state (eg: monomeric, oligomeric, fibrillar) and localisation of intracellular A are believed to be critically important factors. In order to elucidate potential cellular mechanisms related to the intracellular aggregation and localisation of A , a fluorescence-based reporter of A 1-42 misfolding (Chapter 3) was transformed into the Saccharomyces genome knockout collection. Individual strains from this collection were screened for the appearance of A 1-42-GFP fluorescence (indicative of altered A 1-42 folding) and its intracellular localisation. It is believed that at this time, no
105 other group has attempted such a comprehensive survey of genetic factors affecting the aggregation and localisation of intracellular A peptide.
94 single-gene knockout mutants were identified which resulted in the appearance of of A 1-42-GFP fluorescence. Although this set of mutants contained representatives of a diverse range of cellular processes, a relatively compact number of functional categories were found to be statistically over-represented when compared to the screened collection as a whole. These categories included the TCA cycle, regulation of gene expression (via transcriptional activation, repression, or chromatin remodelling), assembly of ETC complexes, and MAPK signalling pathways. Despite falling below statistically over- represented levels, cohesive sets of genes were also identified with roles in phospholipid synthesis and metabolism, mitochondrial housekeeping functions, mitosis, methionine metabolism and the ubiquitin / proteasomal system. In addition to confirming the involvement of processes previously known to be related to A 1-42 pathobiology, the results of this screen suggest the involvement of previously unrealised pathways.
4.3.1: Comparison to genome-wide screens in S. cerevisiae As discussed in Section 1.4.1, the Saccharomyces genome knockout collection has been previously exploited for the identification of gene deletions which enhance in the toxicity of exogenously expressed huntingtin (Huntington's Disease) and -synuclein (Parkinson's Disease). (Willingham, et al., 2003). Intracellular protein misfolding and aggregation is a common feature of HD, PD and AD (Table 1.1). Comparison of mutants identified in fluorescence and toxicity screens may therefore reveal shared or distinct mechanisms related to aggregation and toxicity. The results of this comparison are summarised below in Table 4.3:
Table 4.3: Comparison of mutants identified in the A 1-42-GFP fluorescence screen versus genome-wide -synuclein and Huntingtin screens conducted by Willingham et al (2003)
Screen comparison Common genes
- 6 "# .,- 6 , 9 .,- 6 3& 710 1
106
As noted by Willingham et al (2003), the minimal overlap between mutants enhancing - synuclein and Huntingtin toxicity most likely indicates that these proteins exert toxicity in yeast via independent mechanisms. While a similarly low number of co-occurring mutants were present in the Huntingtin and A 1-42-GFP screens, a number of genes involved in processes common to both experiments were identified.
Two genes identified in common with the -synuclein screen (INO4 and OPI3) also lead to dramatic changes in the localisation of A 1-42-GFP. As described in Sections 4.2.5 and 4.2.6, deletion of either of these genes leads to the appearance of high levels of A 1-42- GFP fluorescence, in a distinctive perinuclear localisation. As INO4 and OPI3 are involved in the transcriptional regulation and synthesis of phospholipids respectively, this may indicate that these systems play a shared role in mediating the behaviours of -synuclein and A . It is important to note that all mutants identified by Willingham et al (2003) in the huntingtin and -synuclein expression screens resulted in severely reduced fitness or lethality, whereas no significant toxicity was observed due to A 1-42-GFP expression in any strain. It should also be pointed out that in this laboratory, expression of -synuclein in the opi3 mutant did not result in significantly enhanced toxicity, although did dramatically affect the localisation of -synuclein (Section 5.2.7). Nonetheless, the specific genes and related processes which are shared among the three screens may be indicative of the diverse range of mechanisms which respond to, or are affected by the presence of these aggregation-prone disease associated proteins.
4.3.2: Screens in Drosophila: involvement of chromatin remodelling and transcriptional regulation
Advances in genetic techniques, such as the creation of C. elegans RNAi gene-knockdown and D. melanogaster EP-insertion mutant libraries have facilitated the application of Saccharomyces-style genome-wide screens in these model organisms. A recent publication described the screening of 1963 individual insertion-mutant Drosophila strains, expressing a secreted form of the A 1-42 peptide under the control of an eye-specific promoter (Cao, et al., 2008). The expression of A 1-42 in the eye led to an age and dose dependent rough
107 eye phenotype, allowing suppressors and enhancers of A 1-42 toxicity to be easily detected. A number of genes involved in processes with known associations to AD pathology were identified via this assay, including genes related to the secretory pathway and cholesterol homeostasis. Additionally, a group of genes involved in various aspects of chromatin remodelling and transcriptional regulation were detected (Sin3A, Rpd3, HDAC4, and SAP130). A number of mutants belonging to this functional group were also found to be significantly over-represented in the A 1-42-GFP fluorescence screen (Table 4.1). While no direct homologs of the Drosophila genes were found in the Saccharomyces fluorescence screen, a mutant encoding an interaction partner of the Sin3p/Rpd3p complex, ume6 , was identified. Ume6p is a zinc cluster DNA binding protein, involved in the transcriptional regulation of a number of early meiosis genes, in response to nutritional cues from the external environment, forming a complex with Sin3p/Rpd3p to deacetylate histones H3 and H4. Taken together with the number of mutants involved in aspects of chromatin structure and regulation which lead to the appearance of A 1-42-GFP fluorescence (Table 6.1), this suggests that the A peptide may interfere in the core mechanisms of gene regulation.
Indeed, Cao et al (2008) hypothesize that high levels of intracellular soluble A 1-42 peptides may titrate out components of the systems which regulate chromatin state and transcriptional control, leading to a cascade of dysregulation across a range of other cellular processes. This hypothesis is supported by the finding that A binds to histone H1 (Duce, et al., 2006) and also that soluble A peptides physically interact with DNA (Barrantes, et al., 2007). Critically, Cao et al (2008) also confirmed that mutations affecting chromatin structure and transcriptional regulation did not necessarily result in a change in the overall amount of expressed A peptide in the Drosophila eye. Rather, there was an shift towards a higher proportion of the total A pool being in a soluble state. This finding corroborates previous studies in mammalian cells which suggest that soluble oligomeric A peptides represent the most toxic species of the peptide (reviewed in Ferreira, et al., 2007).
Similarly, future experiments should confirm that the A 1-42-GFP fluorescence observed in this screen (especially for those mutants known to be involved in transcriptional regulation) is not simply due to significant increases or decreases in the size of the total 108 intracellular A 1-42-GFP pool. For example, mutations in the SSN2, SSN3, and SRB8 genes, members of the SAGA-ADA complex, have been shown to result in decreased expression of a reporter coupled to the GAL1 promoter (Larschan and Winston, 2005), which was also employed in this study for the inducible expression of A 1-42-GFP. While the transcription of several other genes may be also affected by these mutations, the possibility remains that A 1-42-GFP fluorescence in strains such as ssn2 , ssn3 and srb8 is due to a direct reduction in the efficiency of the GAL1 promoter, with a corresponding fall in A 1-42-GFP concentration. A lower concentration of A 1-42-GFP may fall within the inherent protein folding capacity of the cell, or may result in a reduced rate of A 1-42 aggregation, both leading to higher levels of GFP fluorescence.
Ultrastructural studies of post-mortem brain tissue from AD patients have noted the appearance of heterochromatin clumping in neurons which are immunoreactive for A (Gómez-Ramos and Asunción Morán, 2007; Takahashi, et al., 2002). An interaction of A with elements of the machinery of transcriptional regulation and chromatin may dictate localisation of the peptide to the nucleus. In this study, where various A 1-42-GFP expressing strains were stained with DAPI to visualise the nucleus, it was noted on occasion that cells with strong A 1-42-GFP fluorescence often showed little or no DAPI staining. This was despite mitochondrial DNA being successfully visualised within the same cell, eliminating the possibility that the DAPI had not penetrated that particular cell. Although this evidence is anecdotal, it is suggestive of degradation of the nucleus and nuclear DNA in Saccharomyces cells with a high degree of fluorescent A 1-42-GFP, and may be related to the heterochromatin clumping observed by Gomez-Ramos and Moran (2007)and Takahashi et al (2002) in A -immunoreactive neurons. This anecdotal observation could be confirmed by flow cytometry analysis of a population of A 1-42-GFP expressing yeast cells co-stained with a FACS-compatible nuclear DNA dye. If high A 1- 42-GFP fluorescence is indeed correlated with disappearance of DAPI-staining of nuclear DNA, this should result in the appearance of distinct nuclear DNA positive / A 1-42-GFP negative and nuclear DNA negative / A 1-42-GFP positive populations.
109 4.3.3: Mitochondrial dysfunction and A 1-42-GFP fluorescence A significantly over-represented proportion of mutants identified in the A 1-42-GFP fluorescence screen have disrupted mitochondrial function, which is indicated by their respiratory incompetent phenotypes (Table 6.1; Section 4.2.3). This collection of mutants include sub-clusters involved in the assembly of electron transport chain (ETC) complexes, mitochondrial genome maintenance, and the TCA cycle. Mitochondria have long been implicated in the pathobiology of AD (reviewed in Atamna and Frey, 2007). Substantial reductions in the activity of members of the TCA cycle and ETC, such as -ketoglutarate dehydrogenase (Ko, et al., 2001) and complex IV (Cottrell, et al., 2001; Maurer, et al., 2000) have been observed in the brains of AD patients. Additionally, diminished energy metabolism and glucose uptake in the brains of AD patients has been reported (de Leon, et al., 1983), indicating a key role for the mitochondria and energetics in the pathology of AD.
As discussed in Section 4.2.2, A 1-42-GFP fluorescence was detected in several mutants involved in the assembly of components of the ETC, including complex III (cbp3 ), complex IV (cox16 , pet117 , and dubious ORF YDR230W, which overlaps cox20 ), and ATP synthase (atp11 and stf2 ). While these mutations will diminish or abolish the operation of the mitochondrial ETC, it is not known whether the corresponding drop in energy production was related to a change in A 1-42 folding state required to yield GFP fluorescence. When supplied with a fermentable carbon source (such as the 2% w/v galactose utilised in this study), S. cerevisiae preferentially generates energy via glycolysis (fermentation) while growing in exponential phase, which is less dependent on mitochondrial ETC function. As mutant strains were examined for A 1-42-GFP fluorescence when cultures were growing in exponential phase (ie: at an approximate OD600 of 1), it could be expected that energy metabolism was occurring predominantly via fermentation. Thus, energy hypometabolism due to mitochondrial ETC defects are unlikely to be the underlying cause of the changes in A 1-42-GFP folding and fluorescence observed in this study.
An alternative explanation is that a metabolite which is either consumed, produced or otherwise involved with the components of the ETC or TCA cycle may itself affect A 1-
110 42-GFP folding. For example, it has been hypothesized that decreases in complex IV and mitochondrial function may lead to a deficit of heme in the brains of AD patients (Atamna et al, 2002; Atamna 2006). As succinyl-CoA, produced by -ketogluratate dehydrogenase complex (see Figure 4.1) is utilised as a precursor in heme synthesis, defects in TCA enzymes may also deplete reserves of this molecule.
The large proportion of respiratory incompetent strains (over one-third of all mutants displaying A 1-42-GFP fluorescence) and the statistically significant over-representation of mutants involved in the TCA cycle and the assembly of ETC complexes suggests a possible physical interaction of A and the mitochondria. Although detection of mitochondrial co-localisation was not an explicit aim of the A 1-42-GFP fluorescence screening experiment, no obvious signs of this localisation were observed or noted, even in mutant strains which were respiratory incompetent or related to mitochondrial function. This was also true in cases where specific strains were selected for more intensive microscopic examination and stained with fluorescent dyes such as DAPI which reveal not only the nuclear DNA but also that of the mitochondria. These observations do not preclude the localisation of A 1-42-GFP to mitochondria, and additional experiments should be performed with additional agents to visualise the mitochondria, such as the commercially available dye MitoTracker RedTM (Invitrogen), especially in mitochondrially- associated mutants. Given the well documented associations of mitochondrial dysfunction with AD described above, a secondary screen of the A 1-42-GFP-transformed Saccharomyces genome knockout collection (especially for those strains exhibiting A 1- 42-GFP fluorescence) for changes in mitochondrial morphology, respiratory incompetence or production of ROS would potentially reveal additional pathologically-relevant phenotypes associated with the presence of intracellular A .
4.3.4: Phospholipid metabolism and lipid droplets Disturbance of lipid metabolism, particularly of that related to PC synthesis, resulted in the appearance of A 1-42-GFP fluorescence in a distinctive punctate perinuclear pattern (Sections 4.2.5 and 4.2.6). Co-expression of Erg6p-mDsRed, a fluorescent lipid droplet marker protein (Figure 4.10) and staining with the neutral lipid specific dye LipidTOX
111 RedTM (Figure 4.11) provided evidence that the perinuclear A 1-42-GFP puncta were closely associated with lipid droplets. Immunoelectron microscopy studies of post-mortem brain samples from AD patients have also revealed a close physical association of intraneuronal A with lipid droplets (Gómez-Ramos and Asunción Morán, 2007). In this study, all A -immunopositive neurons contained lipid droplets, and the degree of A - immunoreactivity showed a strong positive correlation with the number of lipid droplets per cell. There are striking similarities in the appearance of neuronal and yeast lipid droplet-associated A , with both cell types sharing the same distinctive perinuclear crescent or halo pattern. Gomez-Ramos and Moran (2007) hypothesize that lipid-droplet associated A may have arisen from the lysis of A -containing endosomes or lysosomes. Although the A -1-42-GFP fusion utilised in this study did not possess a targeting sequence which would conceivably translocate it to the endosomal/lysosomal system, the presence of such a highly similar lipid droplet association in both AD neurons and PC mutant yeast strains suggests the existence of a fundamental interaction between lipid droplets and A .
The nature of the interaction between lipid droplets in either neurons or Saccharomyces and the A peptide is not immediately apparent. Lipid droplets have been traditionally considered a relatively static intracellular store of lipids for use in energy generation. More recent studies have revealed lipid droplets to be highly dynamic organelles, being decorated with a functionally diverse range of proteins involved in not only synthesis, storage and utilisation of lipids, but also as a developmentally regulated store of proteins (Cermelli, et al., 2006), a potential site of protein refolding during heat shock (Jiang, et al., 2007), as a convergence point for proteasomal and autophagic degradation (Ohsaki, et al., 2006), and as a possible interface for the intra-organellar trafficking of lipids and proteins (Liu, et al., 2007; Ozeki, et al., 2005). Thus, a number of cellular processes could potentially be related to the appearance of A 1-42-GFP fluorescence in phosphatidylcholine mutants. For example, it is known that expression of A 1-42-GFP in yeast induces a heat shock response (Caine, et al., 2007). Given the apparent role of the lipid droplet as a repository for possibly deleterious proteins (Cermelli, et al., 2006), and as a possible site of post-heat shock protein refolding (Jiang, et al., 2007), the appearance of A 1-42-GFP fluorescence in close association with lipid droplets may represent an attempt by the cell to sequester, solubilise
112 or refold the A peptide. Protein pull-down experiments, using A 1-42-GFP as a bait, could be performed on PC mutant cells to determine if heat shock proteins (or other lipid droplet associated proteins) interact with the A peptide and possibly explain the appearance of GFP fluorescence in these mutants.
Alternatively, the appearance of A 1-42-GFP fluorescence in PC synthesis mutants may be more directly related to the altered composition of phospholipid membranes in these cells. The phospholipid monolayer of lipid droplets in comprises approximately 50% PC (Bartz, et al., 2007). In cho2 and opi3 mutants grown without an exogenous source of choline, intracellular PC levels are significantly depleted (Summers, et al., 1988; McGraw and Henry, 1989). Presuming the phospholipid composition of the lipid droplet monolayer is generally reflective of the general cell-wide changes in phospholipid composition, it is likely that under these conditions the membrane of the lipid droplet would shift towards a higher proportion of PE and PS. Such a change in the fundamental properties of the predominant phospholipid headgroup would likely impact the association of certain proteins with the lipid droplet monolayer. The interaction of A 1-42-GFP with PC-depleted lipid droplets may therefore be via a direct interaction with the monolayer membrane, or via a secondary interaction with another protein whose interaction with the monolayer is altered.
Interestingly, the proportion of PC in the membranes of erythrocytes isolated from AD patients was found to be significantly decreased compared to healthy age-matched control patients (Selley, 2006). Conversely, PE levels were found to be significantly decreased in erythrocyte membranes from AD patients. In addition to changes in phospholipid levels, increased circulating levels of S-adenosylhomocysteine (SAH) were found in the blood of AD patients versus control patients. SAH is produced during enzymatic methylation reactions which utilise SAM as the methyl-group donor and also acts as a feedback inhibitor of these enzymes (see Figure 4.8; the human Cho2p/Opi3p homolog of PEMT is also inhibited by SAH). Thus, it has been hypothesized that the inhibitory effect of SAH on PEMT may be responsible for the reduced PC and increased PE levels observed in erythrocyte membranes (Selley, 2006). This is analogous to the appearance of A 1-42-
113 GFP fluorescence in the ino2 , ino4 , cho2 and opi3 mutants described in this study, and suggests a link between the phosphatidylcholine / phosphatidylethanolamine content of cellular membranes, the state of intracellular A , and the progression of AD.
Notably, the behaviour of a GFP-tagged form of -synuclein, another aggregation-prone protein associated with neurodegeneration was also found to be altered when expressed in Saccharomyces mutants defective in PC synthesis (Section 5.2.7). In wild-type yeast, - synuclein is normally trafficked to the plasma membrane, later becoming localised to multiple small intracellular vesicles (Soper, et al., 2008). However, in cho2 and opi3 mutants, the majority of -synuclein-GFP is found surrounding the LipidTox Red stained core of lipid droplets, presumably localised to droplet phospholipid monolayer. (Section 5.2.7). In a similar manner to the disappearance of A 1-42-GFP fluorescence after supplementation with the Kennedy pathway substrates PDME or choline, addition of these compounds to cho2 and opi3 mutants expressing -synuclein-GFP results in a reversion of -synuclein localisation to the plasma membrane (Section 5.2.7). This result is strongly suggestive of an interaction between the phospholipid composition of membranes, association directly with or near to the lipid droplet, and the cellular response to misfolded or aggregation-prone proteins.
It is not known if the appearance of punctate A 1-42-GFP fluorescence in close association with lipid droplets occurs only in mutants affected in phosphatidylcholine synthesis or if this localisation represents a general case, with only the appearance of fluorescence being specific to these mutants. Immunofluorescence microscopy using anti-A or anti-GFP antibodies could be performed on wild-type cells expressing A 1-42-GFP, or mutant cells supplemented with choline (Figure 4.9) to determine the location of the fusion protein, even when in a misfolded non-fluorescent state. Alternatively, an amyloid-specific dye such as thioflavin T may be used to determine the intracellular location of non-fluorescent A 1-42- GFP. Thioflavin T has traditionally been considered to be an indicator of the fibrillar insoluble form of A . Recent studies have also described the use of thioflavin T analogs for the detection and visualisation of soluble oligomeric forms of A (Maezawa, et al., 2008). Interestingly, cultured neurons and neurons isolated from the brains of 3xTg mice 114 stained with these thioflavin T analogs displayed a crescent-shaped perinuclear distribution of A puncta virtually identical to that observed in cho2 and opi3 mutants. However, no attempt was made by Maezawa et al to further define the intracellular localisation of these puncta, and so it is not known whether they are also closely associated with lipid droplets, or another organelle such as the Golgi or ER.
Further characterisation is required to unequivocally confirm the intracellular localisation of A 1-42-GFP. As mentioned in Section 4.2.6, lipid droplet biogenesis is believed to occur via budding from the cytoplasmic leaflet of the ER membrane. Therefore, lipid droplets can be expected to be found in close association with the ER, and canonical lipid- droplet resident proteins such as Erg6p may also be found in the ER membrane prior to lipid droplet budding. Indeed, transmission electron microscopy (TEM) imaging of lipid droplets have demonstrated a virtually seamless transition from ER to droplet, with ER- derived membranes enveloping the droplet, with ribosomes actually appearing within the lipid droplet core (Wan, et al., 2007). A similarly close association has been described between lipid droplets and peroxisomes, with the two organelles frequently located immediately adjacent to each other (Binns et al, 2006). In certain cases, peroxisomes were even observed to extend processes directly into the core of lipid droplets. To resolve potentially confounding interactions with other organelles, a number of confirmatory experiments should be conducted in the future. Work is currently underway in this laboratory to acquire TEM images of wild-type, cho2 and opi3 strains, each expressing the A 1-42-GFP fusion. Fluorescence microscopy is also being performed using a mDsRed-tagged peroxisomal marker protein. It is anticipated that this additional microscopic characterisation will provide unambiguous evidence for the localisation of A 1-42-GFP within the cell. In addition to microscopy, confirmation of lipid droplet localisation via subcellular fractionation, and A 1-42-GFP detection via western blotting should be performed. This would provide independent biochemical corroboration of any localisation assigned via microscopy.
115 4.3.5: A single protease results in A 1-42-GFP fluorescence The cym1 (YDR430C; also known as MOP112) mutant exhibited punctate A 1-42-GFP fluorescence. Cym1p is a member of the pitrilysin family of metalloproteinases, and is normally located in the mitochondrial intermembrane space. In this localisation, it degrades presequence peptides after their cleavage from imported proteins. It is also responsible for the destruction of misfolded mitochondrial proteins (Kambacheld, 2005). The human homolog of Cym1p, encoded by PITRM1, has been shown to cleave the A 1- 40, A 1-42 and A 1-42 Arctic mutant peptides in at least 7 positions (Falkevall, 2006). While the possibility that A 1-42-GFP may be a Cym1p substrate cannot be completely discounted, the lack of A 1-42-GFP fluorescence in wild-type cells does not appear to be due to partial or complete degradation of the fusion protein (Section 3.2.5). In addition, the A 1-42-GFP ORF does not contain a mitochondrial targeting sequence, and there is no firm evidence of A 1-42-GFP mitochondrial localisation in the numerous fluorescence micrographs obtained throughout this study. It may be possible that Cym1p also exhibits extramitochondrial localisation and activity, although there is no biochemical evidence to support this hypothesis (Kambacheld, 2005).
A more likely explanation for the appearance of A 1-42-GFP fluorescence in cym1 mutants is that lack of wild-type Cym1p activity results in mitochondrial dysfunction, with pleiotropic effects on A 1-42-GFP misfolding and fluorescence. Although the Saccharomyces Genome Database does not note a respiratory incompetent phenotype for cym1 , it has been shown to grow poorly on media containing non- fermentable glycerol as the sole carbon source (Kambacheld, 2005). This is consistent with the significantly over-represented proportion of respiratory incompetent mutants identified in the A 1-42-GFP screen, and further reinforces the idea that defects in mitochondrial function are closely related to the folding of A 1-42.
4.3.6: Future directions The aim of this study was to identify potential genetic factors influencing the misfolding and aggregation of the A 1-42 peptide, in the context of a model eukaryotic cell. To achieve this aim, the A 1-42-GFP fluorescent reporter fusion was expressed in the cytosol
116 of S. cerevisiae. While this approach provides an accessible system to assess a wide range genetic effects on intracellular A 1-42-GFP folding and localisation, it does not attempt to model all relevant aspects of A 1-42 pathobiology. An obvious exclusion from the model is the direct translation of the A 1-42 peptide itself, rather than attempting to reconstitute a functional APP / secretase system in vivo (see Section 1.2.4). Elements of this pathway have been previously studied in S. cerevisiae, including the demonstration of endogenous secretase processing of APP (Zhang, et al., 1994), the reconsitution of -secretase activity by co-expression of human presenilin, nicastrin, APH-1 and PEN-2 (Edbauer, et al., 2003) and the study of the role of the proteasome in degradation of the APP C99 fragment (Sparvero, et al., 2007). Thus, the potential exists to expand the scope of the S. cerevisiae system to include the expression of APP and secretase components to study genetic effects on production, trafficking and aggregation of A on a large scale.
As discussed in Section 1.2.4, processing of APP to produce A most likely occurs within the secretory system and at the plasma membrane. Intraneuronal A is commonly found within organelles related to the secretory or endosomal system, such as the ER, Golgi and multivesicular bodies (MVBs) (reviewed in Laferla, et al., 2007), and it is hypothesized that the intraneuronal toxicity of A may be effected through disruption of these cellular systems. Thus, it may be preferable in future experiments to express a form of the A 1-42- GFP fusion which contains a secretion signal, directing it into the more physiologically relevant environment of the secretory pathway. Such localisation may result in the production of severe toxicity in yeast, which is obviously lacking in the cytosolically expressed form.
The execution of a genome-wide screen of A 1-42-GFP fluorescence and localisation was obviously a labour-intensive, time consuming process. As discussed in Chapter 3, the use of a GFP fusion and the subsequent use of fluorescence as an indication of protein misfolding allows the application of more automated, "shotgun" style techniques with the potential to greatly improve throughput and reduce operator involvement. Each mutant in the Saccharomyces gene deletion collection was produced by the targeted insertion of a cassette into a specific gene, via homologous recombination. In addition to an antibiotic
117 resistance gene (KanMX) to facilitate selection of successful integrants, each insertion cassette also contains a sequence tag which uniquely identifies each mutant. The presence of a uniquely identifying tag integrated into the genome of each mutant allows mixed "one- pot" style experiments to be conducted, whereby large heterogenous populations of mutants are subjected to an experimental treatment simultaneously, and after selection via an appropriate method, mutants of interest are identified by the use of specific PCR primers or by DNA sequencing. Oligonucleotide microarray chips have been produced, containing the entire set of complementary sequences to the yeast deletion tags. These chips may therefore be used to rapidly identify which members of a pooled collection of knockout strains are present in a given mixed population.
An example of how this approach may be applied for screening the gene deletion collection for A 1-42-GFP fluorescence is summarised in Figure 4.14. In this approach, all mutants are pooled and transformed in a "one-pot" experiment. After selection in the appropriate liquid amino acid drop-out media, expression of the A 1-42-GFP fusion is induced via addition of galactose. Mutants which exhibit A 1-42-GFP fluorescence may then be selected via sorting of the pooled mutant collection using FACS (for example, successful differentiation between fluorescent 1-40-GFP and non-fluorescent 1-42-GFP expressing cells was demonstrated in Section 3.2.4). Multiple rounds of subculture and FACS re-sorting may be performed to reduce background levels of non-fluorescent cells, or to sub-sort mutants displaying differing levels of A 1-42-GFP fluorescence into different pools. The final stage is identification of the mutants in the sorted population. Genomic DNA is extracted, PCR amplified and labelled, and used to probe the Saccharomyces deletion tag oligonucleotide chip. Such an approach would substantially reduce the amount of time and labour required to complete a genome-wide assessment of 1-42-GFP fluorescence (or any protein which is amenable to assay of misfolding via inhibition of GFP fluorescence). It would also allow secondary experiments such as treatment of the mutant collection with drugs or chemicals which may influence 1-42-GFP folding to be conducted. Unfortunately in the context of this study, supplies of the commercially prepared oligonucleotide tag-array chips were not available and so the experiments described above were not attempted.
118
pUG35GAL1/A 1-42 GAL
Pool and grow S. cerevisiae Transform pooled strains Induce expression of gene knockout collection with A 1-42-GFP reporter A 1-42-GFP in construct transformed cells
genomic DN A FACS
fluorescent non-fluorescent
Extract, process and probe Subculture sorted strains tag-array chip with genomic and re-sort if necessary DNA to identify mutants
Figure 4.14: Flow chart of potential design for a high-throughput "one-pot" experimental method for screening mutants for A 1-42-GFP fluorescence using FACS and oligonucleotide array chips.
Screening of gene overexpression libraries in yeast models of neurodegeneration has proven fruitful, with the discovery and validation of a mammalian Rab1 homolog which is capable of reversing -synuclein mediated toxicity in S. cerevisiae, C. elegans and mammalian cell culture models (Cooper, et al., 2006). Screening of an overexpression library for genes which modulate 1-42-GFP fluorescence and localisation would provide a valuable complement to the gene knockout screen described in this chapter. The creation of a human brain cDNA library suitable for inducible co-expression in yeast with the 1- 42-GFP reporter was undertaken for this thesis, although time constraints precluded its intended use. The type of screen envisaged may be performed in a similar manner to that summarised in Figure 4.14. Briefly, the human cDNA library may be transformed into
119 cells already bearing the 1-42-GFP reporter construct in a “one-pot” reaction. After selection, double-transformants would have expression of 1-42-GFP and the human cDNA library induced, and then cells exhibiting GFP fluorescence rapidly sorted via FACS. These cells would then be isolated, cultured, and plasmid DNA extracted and sequenced to identify over-expresseed human genes giving rise to altered 1-42-GFP folding.
Key proteins involved in neurodegeneration, such as A , tau and -synuclein are believed to interact both indirectly and directly with each other in the progression of disease states (Rapoport, et al., 2002; Mandal, et al., 2006). As the S. cerevisiae genome does not encode homologs of any of these proteins which could potentially confound experimental analysis, it offers a useful system in which to study their interactions. As discussed in Section 1.4.3, tau has been expressed in S. cerevisiae, and certain aspects of its AD-related behaviour have been recapitulated, including hyperphosphorylation, adoption of a pathologically associated conformation, and the formation of tangles in vitro (Vandebroek, et al., 2005). Furthermore, co-expression of tau with -synuclein in yeast leads to an enhancement in - synuclein toxicity, indicating a synergistic interaction between the two proteins (Zabrocki, et al., 2005). To our knowledge, no co-expression studies of tau and A have yet been performed in S. cerevisiae, and development of such a model may help resolve the ambiguity which remains regarding the interplay between these two central molecules in AD. Construction and expression of GFP-tagged forms of wild-type and the disease associated P301L mutant tau was performed over the duration of the work described in this thesis. The key findings of Zabrocki et al (2005) and Vandebroek et al (2005) were confirmed in our system, and initial experiments with co-expression of A and tau are ongoing. It will be of particular interest to examine the effects of / tau co-expression in mutants positively identified in the 1-42-GFP fluorescence screen.
120 5: Effects of environmental risk factors associated with Parkinson’s Disease on a Saccharomyces model of - synuclein pathobiology
5.1: Introduction and aims The identification of -synuclein as the major component of Lewy Bodies from the neurons of Parkinson's Disease patients led to the first known genetic link to the disease (the PARK1 locus), and in the following decade an additional 12 disease-associated loci were identified (reviewed in Thomas and Beal, 2007). Despite the existence of these loci, fewer than 10% of PD cases are known to be linked to a heritable mutation. Earlier discoveries that the neurotoxin 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) and the herbicide paraquat cause PD-like symptoms in humans and animal models triggered a far-ranging search for environmental factors which may contribute to the development of PD. While several potential agents have been identified by epidemiological and toxicological studies (reviewed in Section 1.3.3), none can singularly and unequivocally account for the range of pathologies and symptoms associated with PD. A “multiple hit” hypothesis of PD pathogenesis is therefore emerging, which recognises the likely interplay of a complex range of genetic and environmental risk factors, which ultimately result in the appearance of the disease (reviewed in Section 1.3.8 and in Sulzer, 2007; Carvey, et al., 2006; Cory- Slechta, et al., 2005). Such a hypothesis also highlights the need for effective experimental models which can encompass both known genetic and environmentally related aspects of PD.
As introduced in Section 1.4.2, several recent studies have demonstrated the utility of S. cerevisiae as a cellular model of -synuclein pathobiology. Overexpression of -synuclein in yeast results in toxicity, alteration of phospholipid metabolism, (Outeiro and Lindquist, 2003; Zabrocki, et al., 2005), interference with ER-Golgi trafficking (Cooper, et al., 2006) and triggering of an apoptosis-like response (Flower, et al., 2005). These effects mirror those observed in mammalian models of PD. Certain studies have also examined the effects of suspected synergistic risk factors, including certain oxidants (Flower, et al., 2005) and metal ions (Zabrocki, et al., 2005), which resulted in an exacerbation of - synuclein toxicity. These results suggest that a yeast model of -synuclein pathobiology
121 may be also be suitable for investigating the role of exogenous stressors and their relationship to -synuclein.
Therefore, the aim of the research described in this chapter was to extend the application of existing S. cerevisiae cellular models of -synuclein pathobiology, to include study of the deleterious interactions of environmental risk agents such as oxidants, metals and pesticides with -synuclein over-expression. Selected metals and pesticides were demonstrated to potentiate -synuclein toxicity. Perturbations in the synthesis of phospholipids which were found to alter the behaviour of an amyloid beta-GFP fusion protein (Section 4.2.6) were also examined with respect to -synuclein-GFP, to discover potentially conserved cellular responses to these misfolded and aggregation-prone proteins.
5.2: Results
5.2.1: ROS generating chemicals enhance -synuclein toxicity The involvement of reactive oxygen species (ROS), oxidative stress and oxidative damage form a cornerstone of research into PD pathobiology. The metabolism of dopamine in dopaminergic neurons leads to the generation of H2O2 and other ROS (Graham, 1978), and thus the dopaminergic cells of the substantia nigra may be particularly vulnerable to insults which result in the creation of additional ROS, such as mitochondrial dysfunction, or occupational exposure to metals or pesticides. To determine if the presence of additional ROS increased the toxicity of -synuclein in the S. cerevisiae model, wild-type cells expressing -synuclein (transformed with the 6821 plasmid; see Table 2.5) were exposed to hydrogen peroxide or menadione (a superoxide radical generator) and growth assessed after 24 hours.
Treatment of -synuclein expressing cells with 1 mM hydrogen peroxide led to a statistically significant reduction in growth after 24 hours, compared to vector-only control cells (Figure 5.1 A). Treatment with 5 mM menadione resulted in an almost complete inhibition of growth in cells expressing -synuclein, although it should be noted that at this concentration, the growth of vector-control cells was also inhibited by approximately 30% (Figure 5.1 B). The reduction in growth due to hydrogen peroxide exposure is consistent
122 with results obtained by Flower et al (2005), who also observed significant enhancement of -synuclein toxicity with hydrogen peroxide. To date, there have been no other reports of menadione enhancement of -synuclein toxicity in yeast. Together, these results demonstrate that -synuclein expression sensitises S. cerevisiae cells to oxidative stress associated with both hydrogen peroxide and superoxide.
Figure 5.1: ROS generating agents enhance -synuclein toxcity. Wild-type cells expressing - synuclein were treated with either 1mM hydrogen peroxide (A) or 5mM menadione (B), and growth assessed after 24 hours. Treatment with either ROS generating compound resulted in significant inhibition of growth of -synuclein cells compared to vector-only control cells.
AB 7 * 6
5
4 vector only w.t. -synuclein 3
2
1
0 01 [H O ] (mM) [menadione] (mM) 2 2
5.2.2: Expression of -synuclein leads to an increase in endogenous production of ROS Expression of -synuclein rendered cells more sensitive to the deleterious effects of the ROS-generating compounds menadione and hydrogen peroxide. A potential explanation for this increased sensitivity is that -synuclein expression itself leads to increased levels of endogenous ROS production, compounding the effects of exogenously added oxidative stressors. To test this possibility, wild-type cells expressing -synuclein were stained with the ROS-sensitive dye dihydroethidium (DHE). DHE is itself non-fluorescent, but may be preferentially oxidized by the superoxide radical to form the highly fluorescent compound ethidium, which may be easily detected via fluorescence microscopy. A significantly higher proportion of DHE-stained cells expressing -synuclein exhibited ethidium fluorescence, versus vector-only control cells (Table 5.1). This result indicates that -synuclein expression in S. cerevisiae leads to an accumulation of ROS, especially the superoxide
123 radical. This result is consistent with that described by Flower et al (2005), who reported that staining of S. cerevisiae cells with dihydrorhodamine 123, another ROS-sensitive dye, demonstrated that -synuclein expression lead to a significant increase in intracellular ROS levels.
Table 5.1: Counts of DHE fluorescence for vector only and -synuclein expressing cells. A significantly higher proportion of -synuclein expressing cells display DHE fluorescence (Fisher's exact test; two-tailed p < 1 x 10-4).
No DHE fluorescence DHE fluorescence % DHE fluorescence vector only -synuclein 4" B6B In cells expressing -synuclein, DHE staining was predominantly observed in cortical tubular or thread-like structures, which is indicative of mitochondrial localisation (Figure 5.2). Mitochondria are a major source of ROS within the cell (particularly the superoxide radical), and ethidium fluorescence is dramatically enhanced upon binding to double- stranded DNA (such as mitochondrial DNA). Increased levels of mitochondrial ROS in cells expressing -synuclein versus controls may indicate that -synuclein is capable of interfering with normal mitochondrial function, leading to inhibition of growth. 124 vector only -synuclein light DHE fluorescence light DHE fluorescence Figure 5.2: Expression of -synuclein increases endogenous ROS. Wile-type cells expressing a- synuclein or transformed with an empty vector control were stained with dihydroethidium (DHE), a dye which becomes fluorescent in the presence of ROS. Cells expressing -synuclein displayed higher levels of DHE fluorescence versus vector only controls, indicating presence of higher levels of ROS. Three replicate panels are shown for control and -synuclein expressing cells. 5.2.3: Treatment with the antioxidant compounds ascorbate and - tocopherol does not reduce -synuclein toxicity The observations that ROS-generating compounds enhance -synuclein toxicity (Section 5.1), and that -synuclein expression resulted in an increase in endogenous ROS (Section 5.2) suggested that treatment with antioxidant compounds may reduce -synuclein related toxicity in the S. cerevisiae model. Indeed, various compounds with direct or indirect antioxidant effects have been proposed as Parkinson's Disease therapeutics, including coenzyme Q (Müller, et al., 2003; Horvath, et al., 2003), curcumin (Jagatha, et al., 2008; 125 Pandey, et al., 2008), synthetic SOD/catalase mimetics (Peng, et al., 2005), -tocopherol (Fariss and Zhang, 2003) and ascorbate (Fahn, 1992). To determine if treatment with antioxidants would ameliorate -synuclein induced toxicity, a range of -tocopherol and ascorbate concentrations were added to the growth media of wild-type cells expressing -synuclein, and growth assessed after 24 hours (Figure 5.3). Treatment with 25 μM or 50 μM -tocopherol (in an acetone carrier) did not result in any increase in growth of -synuclein-expressing cells, relative to a vector-only control. Similarly, treatment with ascorbate at 0.5 mM, 1 mM or 10 mM concentrations resulted did not lead to difference in the yield of -synuclein-expressing cells, relative to the vector- only control. There are a number of potential explanations for the lack of protective effect afforded by - tocopherol and ascorbate treatment. The concentrations of -tocopherol and ascorbate used in this experiment may not have been sufficiently high to provide an effective antioxidant supplement. However, a minor, yet statistically significant decrease (p < 0.01) in growth was observed in -synuclein-expressing cells when treated with the highest concentration of ascorbate (10 mM) used in this experiment. Another explanation for the lack of effect is that the increased sensitivity to ROS-creating agents (Section 5.1) and the increases in endogenous ROS in -synuclein-expressing cells (Section 5.2) may be downstream consequences of the mechanism through which - synuclein exerts its toxicity in yeast. Effective reductions in the levels of ROS by ascorbate and -tocopherol may therefore not necessarily result in an increase in growth in - synuclein expressing cells. 126 vector only w.t. -synuclein 6 7 ab 5 6 5 4 4 3 3 2 2 1 1 0 0 0 0.5 1 10 25 50 [ascorbate] (mM) -tocopherol ( μM) Figure 5.3: Treatment with antioxidants does not reduce -synuclein toxicity. OD(600) measured after 24 hours growth of vector only or -synuclein expressing wild-type cells, treated with ascorbate (a) or -tocopherol (b). Treatment with either antioxidant does not result in an increase in growth of -synuclein expressing cells. 5.2.4: The pesticide rotenone does not enhance -synuclein toxicity Rotenone (discussed in Section 1.3.6) is a lipophilic compound derived from plants, and is used commercially as a pesticide. It is capable of producing Parkinson's Disease-like symptoms, such as selective loss of dopaminergic neurons and accumulation of - synuclein-positive inclusions when chronically administered to rats (Betarbet, et al., 2000) as well as in human neuronal cell culture (Sherer, et al., 2003). In these mammalian models of Parkinson's Disease, rotenone inhibits the activity of complex I of the mitochondrial electron transport chain, leading to death of cells in the vulnerable substantia nigra. In vitro experiments have demonstrated that micromolar concentrations of rotenone are capable of altering the conformation of -synuclein, and accelerating its rate of fibirillization, and it has been proposed that direct interaction of rotenone with -synuclein may provide an alternative explanation to account for the appearance of Parkinson's-like symptoms in rotenone models (Uversky, et al., 2001). S. cerevisiae provides a useful in vivo model to 127 test this hypothesis, as its mitochondrial electron transport chain lacks a complex analogous to complex I of mammalian cells, rendering it resistant to rotenone toxicity. It is therefore possible to use S. cerevisiae to study the effects of rotenone on -synuclein toxicity, independently of any confounding involvement of complex I inhibition. Exposure of change in growth yield after 24 hours (Figure 5.4). Additionally, rotenone exposure did not result in any change in -synuclein-GFP localisation when compared to control cells exposed to DMSO carrier alone, or growing in normal DMSO-free media (data not shown). Thus rotenone exposure does not result in complex I-independent - synuclein toxicity, or any discernable in vivo change in localisation at concentrations of up to 250 μM. It may be possible that an effect could be observed at higher concentrations of rotenone. However, as rotenone is highly lipophilic, it was difficult to keep the compound in solution in SC medium without the addition of higher amounts of DMSO carrier which in itself results in reduction in growth, even in cells bearing only the empty vector control. vector only w.t. -synuclein [rotenone] ( μM) Figure 5.4: The pesticide rotenone does not enhance -synuclein toxicity. Wild-type cells expressing -synuclein were exposed to increasing concentrations of rotenone in s 5% DMSO carrier. No differences in growth were detected compared to the vector-only control at any concentration tested. 128 5.2.5: The pesticide dieldrin enhances -synuclein toxicity Organochlorine pesticides are among the environmental risk factors proposed to play a role in the development of Parkinson's Disease (reviewed in Hatcher, et al., 2008). Although they are now banned in many countries, organochlorine pesticides such as dichlorodiphenyltrichloroethane (DDT) and dieldrin have been widely utilised in agriculture, and are highly persistent in the environment. A comprehensive meta-analysis of the published reports of the relationship between PD and pesticide exposure concluded that a generic link between the two indeed exists (Brown, et al., 2006). In particular, higher levels of dieldrin have been detected in the post-mortem brains of Parkinson's Disease sufferers, versus non-affected control brains (Fleming, et al., 1994). Dieldrin has also been demonstrated to produce effects such as increased oxidative damage (Hatcher, et al., 2007), inhibition of proteasomal activity (Sun, et al., 2005), and alteration of the dopaminergic system (Richardson, et al., 2006), all of which may be directly related to its hypothesized role in the development of Parkinson's Disease. To determine if the S. cerevisiae expression system might be useful to further understand the role of organochlorine pesticides in Parkinson's Disease, wild-type cells expressing both wild-type and A53T mutant forms of -synuclein were exposed to dieldrin, and their growth measured after 24 hours. Exposure to 250 μM dieldrin (in 5% DMSO carrier) resulted in statistically significant reductions in growth of cells expressing both wild-type -synuclein and A53T -synuclein (Figure 5.5). The growth reduction was greater for A53T -synuclein, and indicates this mutant -synuclein selectively sensitises cells to the toxic effects of dieldrin. No reduction in growth was observed in vector-only control cells exposed only to a 5% DMSO carrier. While there are little data regarding the effects of organochlorine pesticides on S. cerevisiae, dieldrin exposure results in increased ROS production in murine microglial cell culture (Mao, et al., 2007) and in dopaminergic neurons of whole mice (Hatcher, et al., 2007). To determine if dieldrin exerted similar effects in S. cerevisiae, cell lacking the superoxide dismutase gene SOD1 were exposed to dieldrin. No growth could be detected in sod1 mutant cells exposed to 250 μM dieldrin after 24 hours (data not shown). Since 129 sod1 mutants are highly sensitive to oxidative stress, this result indicates that dieldrin enhancement of -synuclein toxicity may also be effected through a ROS-related mechanism, specifically one involving the superoxide radical. 5% DMSO 5% DMSO + 250 μM dieldrin * p < 0.003 ** p <= 1 x 10-4 vector only w.t. -synuclein A53T -synuclein Figure 5.5: Dieldrin enhances -synuclein toxicity. Wild-type cells expressing wild-type or A53T mutant -synuclein were treated with 250mm dieldrin (in a 5% DMSO carrier) and growth assessed after 24 hours. Dieldrin significantly inhibited growth of -synuclein expressing cells versus the vector only control. Growth inhibition was greater for the A53T mutant than for wild-type - synuclein. 5.2.6: Aluminium enhances -synuclein toxicity Epidemiological analyses have highlighted exposure to metals including iron, manganese, copper and lead as potential environmental risk factors leading to early onset of PD (Gorell, et al., 1999; Gorell, et al., 2004). The detection of an accumulation of aluminium in the substantia nigra of PD sufferers (Hirsch, et al., 1991; Good, et al., 1992) has lead to the proposal that aluminium may also contribute to the development of idiopathic PD (Altschuler, 1999). In vitro studies demonstrate that aluminium interacts directly with - synuclein and accelerates its rate of fibril formation (Uversky, 2001). The aluminium- mediated fibrilization of may -synuclein is also synergistically enhanced by the presence of pesticides (Uversky, et al., 2002). In vivo, aluminium has been demonstrated to inhibit 130 key enzymes in the tricarboxylic acid cycle, leading to impaired ATP production (Mailloux, et al., 2006). To test for an interaction between aluminium and -synuclein in the S. cerevisiae model, cells expressing wild type, A53T or A30P forms of -synuclein were exposed to a range of aluminium chloride concentrations and their growth measured after 24 hours. Significant growth reductions were observed for cells expressing wild-type and A53T mutant forms of -synuclein at the lowest concentration of AlCl3 tested (0.5 mM; Figure 5.6). There was also a marked reduction in growth yield in cells expressing the A30P - synuclein mutant. Although A30P mutant -synuclein is associated early-onset Parkinson's Disease (Krüger, et al., 1998), its expression in S. cerevisiae is not associated with toxicity. Additionally, A30P -synuclein does not localise to the plasma membrane or form aggregates, which are traits associated with wild-type and A53T -synuclein expressed in yeast. Thus, aluminium chloride exposure at concentrations in excess of 0.5 mM appears to have unmasked a latent toxicity of A30P -synuclein in yeast. 0 mM AlCl3 0.5 mM AlCl3 1.0 mM AlCl3 1.5 mM AlCl3 vector only w.t. -synuclein A53T -synuclein A30P -synuclein Figure 5.6: Aluminium enhances -synuclein toxicity. Wild-type cells expressing wild-type, A53T or A30P -synuclein were treated with a range of aluminium chloride concentrations, and growth assessed after 24 hours. Aluminium enhanced -synuclein toxicity at all tested concentrations, including for cells expressing the normally non-toxic A30P mutant form. 131 5.2.7: Mutants of the phosphatidylcholine biosynthesis pathway display altered -synuclein-GFP localisation Deletion mutants of the CHO2 and OPI3 genes displayed increased fluorescence, and lipid droplet localisation of an A 1-42-GFP fusion protein (Section 4.2.6). CHO2 and OPI3 encode methyltransferase enzymes, which together catalyse the trimethylation of phosphatidylethanolamine (PE) to form phosphatidylcholine (PC). Without an exogenous source of choline, cells lacking either of these genes are unable to synthesise phosphatidylcholine, which results in the accumulation of methylated phosphatidylcholine precursors and large-scale remodelling of cellular membranes (reviewed in Section 4.2.6 and Figure 4.8). To determine if blocking the cell's ability to synthesize phosphatidylcholine altered the intracellular localisation of -synuclein, wild-type and A30P mutant -synuclein-GFP fusions were expressed in cho2 or opi3 deletion mutants, and examined with fluorescence microscopy. Expression of wild-type -synuclein-GFP in cho2 or opi3 mutants resulted in the appearance of distinctive new localisations (Figures 5.7 B and 5.8 B, respectively). In contrast to the plasma membrane localisation observed in wild-type cells, in cho2 or opi3 mutants the fusion protein was localised to open ring-like structures and in puncta of various sizes. The distribution of these features was in a distinctive semi-circular or crescent arrangement, similar to that observed for A 1-42-GFP in the same strains (Section 4.2.6). Expression of the A30P -synuclein-GFP point mutant in cho2 (Figure 5.9) or opi3 (Figure 5.10) did not result in any change of localisation when compared to wild- type cells, with A30P -synuclein-GFP remaining distributed evenly throughout the cell. Colocalisation studies with either a mDsRed-tagged lipid droplet resident protein (Erg6p), or the neutral lipid stain LipidTox Red, indicated that A 1-42-GFP is closely associated with lipid droplets in cho2 and opi3 mutants (section 4.2.6). To determine if - synuclein-GFP was also localised to lipid droplets in the cho2 or opi3 mutants, the Erg6p-mDsRed fusion protein was co-expressed with -synuclein-GFP. Overlay of the red Erg6p-mDsRed and the green -synuclein-GFP fluorescence images from both 132 cho2 (Figure 5.7 D) and opi3 (Figure 5.8 D) mutants revealed substantial signal overlap, indicative of colocalisation. No colocalisation was observed between A30P - synuclein-GFP and Erg6p-mDsRed in either cho2 (Figure 5.9 D) or opi3 (Figure 5.10 D). cho2 and opi3 mutants are able to synthesise phosphatidylcholine if an external source of choline is available, via the Kennedy salvage pathway (refer to Figure 4.8). To test if the presence of a functioning phosphatidylcholine synthesis pathway in a cho2 mutant resulted in reversion of -synuclein-GFP localisation to that observed in wild-type cells, cho2 cells co-expressing -synuclein-GFP and Erg6p-mDsRed were grown in the presence of 1 mM choline chloride. Addition of exogenous choline resulted in the localisation of wild-type -synuclein-GFP to the plasma membrane in cho2 cells, as per wild-type cells (Figure 5.11). Overlay of the -synuclein-GFP and Erg6p-mDsRed fluorescence channels revealed no signal overlap (Figure 5.11 D), indicating that the association of -synuclein-GFP with lipid droplets observed in unsupplemented cho2 cells was abolished. This choline-promoted abolition of lipid droplet localisation is consistent with that observed for cho2 and opi3 mutants expressing A 1-42-GFP, and may indicate an underlying common mechanism between -synuclein-GFP and A 1-42- GFP localisation in phosphatidylcholine-depleted cells. 133 134 135 136 137 138 5.3: Discussion 5.3.2: Enhancement of -synuclein toxicity by aluminium Aluminium is among the metals identified as playing a potential role in the development of Parkinson’s Disease. As aluminium is not a transition metal, its ionised form is not thought to be capable of participating in ROS-generating redox cycling reactions in an analogous manner to iron and copper ions. Nonetheless, aluminium exposure results in the production of ROS and oxidative stress in systems as diverse as rat brains (Gómez, et al., 2005), plants (Richards, et al., 1998) and yeast (Zheng, et al., 2006). Aluminium is toxic to cells, causing changes in lipid metabolism (Pejchar, et al., 2007), inhibiton of components of the tricarboxylic acid cycle (Mailloux, et al., 2006), as well as the aforementioned generation of ROS and associated oxidative damage to macromolecules. How these forms of toxicity are linked to the progression of Parkinson’s Disease remains unclear. Thus, the applicability of the S. cerevisiae -synuclein expression model was investigated, with repsect to aluminium toxicity. As described in section 5.2.6, treatment of S. cerevisiae expressing -synuclein with AlCl3 resulted in significant reductions of growth when compared to vector-control cells. Almost complete inhibition of growth was observed in -synuclein expressing cells at AlCl3 concentrations (1 mM) which only resulted in a minor (approximately 10%) reduction in growth of cells expressing the vector control. This highlights the potential of aluminium in enhancement of -synuclein toxicity. Interestingly, this strong enhancement was also observed in cells expressing the A30P point mutant form of -synuclein. This -synuclein mutant is associated with early onset of Parkinson’s Disease clinically, but previous studies (Outeiro and Lindquist, 2003) have demonstrated that the A30P mutation virtually abolishes -synuclein toxicity when expressed in S. cerevisiae, and that it does not form the distinctive puncta observed for the wild-type and A53T mutant forms of the protein. It has been hypothesized that the substitution of a structurally-disruptive proline residue in the A30P form leads to an -synuclein with significantly altered behaviour, such as its membrane affinity and conformation, which may be responsible for its dramatically changed behaviour. However, in the presence of aluminium, the lack of toxicity associated with A30P -synuclein is reverted to wild-type levels, in S. cerevisiae at least. The 139 mechanisms by which aluminium causes cellular toxicity and exposes A30P -synuclein toxicity may give an insight into why this mutant causes early-onset Parkinsonism. The enhancement of -synuclein toxicity in S. cerevisiae by aluminium may occur through a number of mechanisms. Aluminium exposure results in the production of ROS in mammalian models, including human neural cell culture (Alexandrov, et al., 2005) and rat brain (Gómez, et al., 2005), as well as in S. cerevisiae (Zheng, et al., 2006). Expression of -synuclein also increases ROS accumulation in yeast, and its toxicity is enhanced by treatment with ROS-generating agents such as hydrogen peroxide and menadione (Section 5.2.1; Flower et al, 2005). Therefore, one possible explanation for aluminium enhancement of -synuclein toxicity may be that the additional burden of aluminium-mediated ROS overwhelms the antioxidant defenses of the cell, leading to oxidative damage and cell death. This hypothesis could be tested by measuring ROS levels in -synuclein-expressing cells after aluminium exposure, using methods such as DHE fluorescence (section 5.2.2). Markers of oxidative damage such as levels of protein carbonylation (an indicator of oxidative damage to proteins) and malondialdehyde (a by-product of lipid oxidation) could also be assessed to determine if synergistic oxidative damage was occurring. In addition to synergistic toxicity from additive levels of ROS, aluminium and -synuclein may interfere with other, potentially overlapping areas of cellular metabolism. Genome- wide knockout screens were performed in S. cerevisiae identified genes which are required for resistance to -synuclein (Willingham, et al., 2003) and aluminium toxicity (Kakimoto, et al., 2005). Both screens independently identified common processes involved in vesicular trafficking and lipid metabolism as being required for resistance to either - synuclein or aluminium toxicity. Additionally in yeast, overexpression of anti-apoptotic genes prevents programmed cell death after high level aluminium exposure (Zheng, et al., 2006), and deletion of a pro-apoptotic gene abolishes -synuclein toxicity (Flower, et al., 2005). Induction of apoptosis in cultured neural cells is a feature common to both Parkinson's Disease (Saha, et al., 2000; Stefanova, et al., 2001) and aluminium exposure (Toimela and Tähti, 2004). Additionally, aluminium exposure leads to neural apoptosis in animal models (Savory, et al., 2003). This overall commonality may indicate that - 140 synuclein and aluminium share overlapping mechanisms or targets of their respective toxicities. Expression of -synuclein inhibits the activity of phospholipase D in yeast (Outeiro and Lindquist, 2003). Phospholipase D, encoded by the SPO14 gene in S. cerevisiae hydrolyses phosphatidylcholine (PC), producing choline and phosphatidic acid (PA) (Waksman, et al., 1996). PA is an important regulator of general phosholipid synthesis, binding to and deactivating the Opi1p transcriptional regulator, itself a repressor of the family of phospholipid synthesis genes regulated by the inositol-choline responsive element (Loewen, et al., 2004). Changes in PA level can therefore alter phospholipid metabolism at a fundamental level. Interestingly, aluminium has also been found to inhibit phospholipase D activity in Coffea arabica cells (Pejchar, et al., 2007). Although not yet examined in S. cerevisiae, it may be possible that -synuclein and aluminium are both capable of influencing PA levels via phospholipase D inhibition, and hence, broadly influencing lipid metablism in the cell with potentially deleterious effects. The observation that -synuclein- GFP undergoes a change in localisation from the plasma membrane to lipid droplets in opi3 mutant cells (section 5.2.7), provides evidence that phospholipid composition and metabolism plays a role in regulating at least a portion of -synuclein behaviour. This may have important implications for the elucidation of the mechanism by which aluminium enhances -synuclein toxicity. To date, synergistic toxicity of -synuclein and aluminium in S. cerevisiae has not been described in the literature. This novel finding lends further support to the hypothesis that aluminium exposure is a contributing risk factor in the development and progression of Parkinson's Disease. The existence of related and overlapping processes in yeast that are involved in both aluminium and -synuclein toxicity highlights the utility of the S. cerevisiae model for the study of environmental factors related to Parkinson's Disease pathology. 141 5.3.3: Environmental factors: enhancement of -synuclein toxicity via metals and pesticides Together with metals, exposure to organochlorine pesticides such as dieldrin has also been identified as a risk factor for the development of Parkinson’s Disease. Despite being banned in the 1980s in most western countries, organochlorine pesticides such as dieldrin remain ubiquitous within the environment due to their high stability. Being lipophilic, dieldrin persists stably in fatty tissues, including the central nervous system, and is degraded in the environment slowly, with a half life of approximately 2.5 years (Hatcher, et al., 2008). Like other stable environmental pollutants, dieldrin concentrates to high levels in organisms at the top of their respective food chains. A striking example of the persistence and bioaccumulation of dieldrin is its detection in human breast milk samples in communities where the use of dieldrin was banned 20 years previously (Mueller, et al., 2008). As discussed in Section 1.3.7, dieldrin has been detected in the post mortem brains of PD patients at levels significantly higher than healthy control patients, and there is convincing epidemiological evidence of an association between organochorine pesticide exposure and the development of PD. The enhancement of -synuclein toxicity by dieldrin in the S. cerevisiae model (Section 5.2.5) may therefore represent a relevant system for the study of pesticide exposure in Parkinson’s Disease. As no inhibitory effect was observed in the growth of vector-only control cells at the 250 μM concentration of dieldrin used in this experiment, the mechanism of toxicity appeared to be highly specific to cells expressing -synuclein. Furthermore, the increased toxicity of dieldrin in cells expressing the A53T mutant compared to the wild-type form of -synuclein is especially interesting, as other external stressors (such as aluminium chloride exposure; Section 5.2.6) did not result in similar relative differences between the two -synuclein forms. A53T -synuclein displays significant differences in its interactions with membranes (Jo, et al., 2004) and in its propensity to self-aggregate (Li, et al., 2001) compared to the wild-type form. This result supports previous findings in transgenic mouse PD models expressing wild type and A53T mutant -synuclein (Norris, et al., 2007). In this case, chronic treatment with the pesticides maneb and paraquat lead to the development of significantly more severe neuronal 142 pathology in mice expressing A53T, compared to wild-type -synuclein. While maneb and paraquat are distinct both from dieldrin, and each other, in terms of their chemical structures and industrial applications, their selective enhancement of A53T -synuclein- related toxicity in both mammalian organismal and yeast cellular models possibly indicates a fundamental interaction between mutant -synuclein and a broad range of pesticides. These interactions and synergies between genetic and environmental risk factors support evolving "multiple hit" hypotheses of PD pathogenesis (Sections 1.3.8 and 5.1), and further demonstrate the utility of the Saccharomyces cellular model as a tool for studying the role of -synuclein in PD. The effects of dieldrin on the physiology of S. cerevisiae do not appear to have been well studied by other groups, and as such it is difficult to propose a confident hypothesis to explain the -synuclein specific toxicity observed here. Exposure of sod1 mutant cells to dieldrin resulted in complete inhibition of growth (section 5.2.5), and -synuclein expression in S. cerevisiae leads to ROS production (section 5.2.2; Flower et al, 2005). In mammalian cells, dieldrin is believed to produce ROS by inhibition of the mitochondrial complex III (Bergen, 1971). Dieldrin treatment results in the production of ROS in rodent microglial cells (Mao, et al., 2007), depletion of glutathione reserves and increase in total protein carbonyls (Hatcher, et al., 2007), and induction of DNA repair responses specific to oxidatively damaged nucleotides in mouse brains (Sava, et al., 2007). Therefore, it may be reasonable to propose that the additional toxic effects of dieldrin on yeast cells expressing -synuclein are mediated by a ROS-related mechanism. If the toxic effects of dieldrin are produced by the same ROS-related mechanisms as in neuronal cells, it ought to be possible to detect the production of these ROS using methods such as DHE staining (section 5.2.2). Dieldrin-induced ROS would also result in oxidative damage to proteins, lipids and DNA, which could be detected by traditional biochemical methods. The toxicity of dieldrin could also be expected to be greatly enhanced in mutants lacking key antioxidant defense enzymes such as Sod1. Further experimentation in this area would be useful in further elucidating the mechanisms by which dieldrin exerts its - synuclein specific toxicity, particularly with regard to the A53T mutant form. 143 5.3.4: Phospholipid synthesis, lipid droplets and -synuclein When expressed in wild-type S. cerevisiae, both wild-type and A53T mutant -synuclein are initially localised to the plasma membrane (Outeiro and Lindquist, 2003; Flower, et al., 2005; Zabrocki, et al., 2005; Sharma, et al., 2006), after trafficking via the secretory pathway (Dixon, et al., 2005). This pool of plasma membrane localised -synuclein acts as a focal point for the formation of small aggregates approximately 80 minutes post- induction. These enlarged aggregates eventually detach from the plasma membrane and distribute throughout the cytosol, in the form of small vesicles (Soper, et al., 2008). This localisation is consistent with the proposed normal function of -synuclein, which is a lipid-associated regulator of vesicle fusion (reviewed in Bonini and Giasson, 2005). As presented in section 5.2.7, disruption of phosphatidylcholine synthesis via deletion of either the CHO2 or OPI3 genes, resulted in a dramatic change in the localisation of -synuclein- GFP. Plasma membrane localisation was abolished, with -synuclein-GFP appearing in a crescent-like distribution of puncta and small open ring-shaped structures (Figures 5.6 and 5.7). This localisation was determined to be to lipid droplets, as evidenced by co- localisation with mDsRed tagged Erg6p, a known lipid droplet resident protein (Athenstaedt, et al., 1999; Binns, et al., 2006), and LipidTOX Red, a fluorescent neutral lipid stain In contrast, localisation of the A30P mutant -synuclein-GFP was unchanged in cho2 and opi3 mutants, remaining distributed throughout the cytosol (Figures 5.9 and 5.10). Lipid droplet localisation of -synuclein has been reported in post mortem brain samples (Halliday, et al., 2005) and in HeLa cells (Cole, et al., 2002). When expressed as a GFP fusion in HeLa cells, the amount of -synuclein lipid droplet localisation increased when the cells were lipid loaded, via addition of oleate to the growth medium. Fluorescence microscopy images of the transfected HeLa cells revealed that -synuclein was present in the phospholipid monolayer of the lipid droplet, arranged around the circumference of its neutral lipid core. This appearance is markedly similar to that observed in the -synuclein- GFP expressing cho2 and opi3 mutants described in section 5.2.7, providing corroborating evidence for the lipid droplet localisation proposed in this thesis. The lack of lipid droplet localisation for the A30P -synuclein mutant expressed in cho2 and 144 opi3 cells was consistent with the localisation reported by Cole et al, and with other reports of decreased membrane affinity of the A30P mutant (Jensen, et al., 1998; Jo, et al., 2002). The effects of CHO2 and OPI3 deletion are discussed in detail in section 4.2.6, and are summarised again here. Briefly, Cho2p and Opi3p act sequentially to produce phosphatidylcholine (PC), via the trimethylation of phosphatidylethanolamine (PE). Disruption of either gene results in the loss of the ability to synthesize PC from PE, an accumulation of methylated PE derivatives (such as phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine), and large scale remodelling of cellular membranes, with associated alterations to cellular metabolism. Although S. cerevisiae is capable of PC synthesis via the alternative Kennedy salvage pathway, this requires the uptake of appropriate precursors (such as choline) from the medium. These precursors were not added to the growth media in the experiments described here unless otherwise noted, thereby forcing the cell to deplete its reserves of PC. Inhibition of PC synthesis in cho2 and opi3 mutants results in the depletion of PC, and consequently large increases in the proportions of PI and PE (Summers, et al., 1988; McGraw and Henry, 1989). In opi3 mutants, the proportion of the partially methylated PC precursors phosphatidylmonomethylethanolamine (PMME) and phosphatidyldimethylethanolamine (PDME) also increases greatly. The most abundant phospholipid in the surrounding monolayer of mammalian lipid droplets is PC (Bartz, et al., 2007). Although the phospholipid profile of S. cerevisiae lipid droplets has not yet been determined, it is likely that PC is also the major component of this monolayer due to the high level of conservation of other lipid droplet characteristics. Therefore, deletion of cho2 and opi3 could be expected to result in depletion of PC from the lipid droplet phospholipid monolayer. It is not known which phospholipids replace PC in the lipid droplet membranes of cho2 and opi3 mutants. The interaction of -synuclein with cellular membranes is influenced by their composition. -synuclein exhibits an increased affinity for membranes containing higher proportions of 145 negatively charged phospholipids (eg: PI, PA, PG, PS) when compared to membranes with higher proportions of neutral or zwitterionic phospholipids (eg: PC, PE) (Madine, et al., 2006; Rhoades, et al., 2006). Thus, the depletion of zwitterionic PC from cellular membranes in cho2 and opi3 mutants, and the corresponding increases in the proportion of anionic PI (Summers, et al., 1988; McGraw and Henry, 1989) may be responsible for the localisation of -synuclein to lipid droplets. The phospholipid composition of lipid droplets purified from cho2 and opi3 mutants would need to be investigated to determine if there was an increase in anionic phospholipid concentation which would support this hypothesis. Additionally, depletion of PC in cho2 and opi3 mutants is accompanied by compensatory remodelling of the fatty acid chains of PE to help cells maintain correct membrane curvature and fluidity (Boumann, et al., 2006). Alterations in membrane curvature are proposed to influence the interaction of -synuclein with membranes (Davidson, et al., 1998; Madine, et al., 2006), and may also explain the changes in -synuclein localisation described here. It is worth noting that changes in fatty acid composition have been reported in both dopaminergic neurons overexpressing - synuclein, and in the brains of patients suffering from -synucleinopathies (Sharon, et al., 2003). These changes in fatty acid composition were linked with alterations in membrane fluidity and curvature. The activity of phospholipid synthetic and remodelling enzymes was also found to be altered in the post mortem brain samples of Parkinson's Disease patients (Ross, et al., 1998). Studies of lipid droplet proteomes in Drosophila and rat have identified heat shock proteins as being present in abundance (Cermelli, et al., 2006; Jiang, et al., 2007). Of particular note is that members of the Hsp70 family of chaperones have been identified. Overexpression of the SSA3 gene in S. cerevisiae, a Hsp70 homolog in yeast, was demonstrated to rescue cells from -synuclein toxicity (Flower, et al., 2005), and the action of Hsp70 has been hypothesized to be a potential target for development of therapeutics for the treatment of Parkinson’s Disease. It is tempting to speculate that the localisation of - synuclein to lipid droplets may represent an attempt by the cell to sequester the aggregation-prone protein into an environment rich in chaperones, where it may be productively folded. Recent studies have revealed a link between lipid droplet protein 146 localisation and their delivery to the proteasome and/or lysosome for degradation (Ohsaki, et al., 2006). It is possible that the lipid droplet represents a collection point for hydrophobic or membrane associated proteins which are misfolded, prior to their delivery to the degradative systems of the cell. 147 6: Appendix Table 6.1: Results of the Saccharomyces genome-wide screen for mutants exhibiting A 1-42-GFP fluorescence. Gene ORF Description A 1-42-GFP Human Respiratory Name Name fluorescent homolog incompetent morphology ? Mitochondrial genome maintenance, protein synthesis and import ! 8* 1 !)*%; 5 < + ; G - ; G # 8.1 & - 5,8; 8 <@+ # 7<774 ++) ( - 8 ( ; G ( 5 8/1 & - 5,8/; 8 + # 7<774 ++) ( - 8 ( ; G ( 5 ! 0 8* % 5 <C 0 !&( $ 8*< % %*1<4; 8 C 1<4 !&(5 8* % 8 <@C " # 8/1 G $) 1; 8 B + ; $) 09 : / & ! 8+* & 5 8 B+ ; -- 5 &! 6 8$* % 8 4 + $6 &B &!5 8H* % 8 <+ $6 &@ "%2 8'* % & )>,%; 8 B@C ); .) ) ( - ? - &6 TCA cycle / respiration / electron transport chain 1 81*< G ( +' ; 8 + 9)+: G ? ; " 851< % G ),; 8 ; 4+ ? ) ; , ,, ) , ( ( 148 81 % G >M++; 8 4C G ( ? G 9 (:; ? + ( ? + +/< 1 8 8H1 % 8 !0 81 5 ( 5 BB+ ( 54< ? 5@ 152 Endoplasmic reticulum # 8!1 ! $* ? !!! 5 K C ; $* Glutatione metabolism '"" 8$1 0? ? 5 @C ; ., Inositol phosphate metabolism , 8*< ! < 4 "0 0 , !E; 5 4+ G ? < 4 " < 4 "(0 9: 0 ? ; 0 - ; 0 - Protein folding / chaperone 9 851 - #& 95H: 5 @@C ; - ( ? N 3O ; Pyridoxine metabolism + 8/* ( 9 ( : 5'; 8 <4+ ( $6 ( 4 %( ( ; ( Retrograde endosome-Golgi transport 7 0 8* & ( -- - 5 4C . &5*$ . . ? 0; ? &( Riboflavin synthesis & 8/1 .) !!; 8 <<+ - ? Ribosome biogenesis '+" 8/* - 0? ; )*1; ) 8 B@C ( ; ., -; ( Transcriptional regulation of nitrogen utilisation ' " 8,1 ) - -- 5 C ; .) 5 ; - > tRNA metabolism + ' 8,1 5 *5J & ,E&.< ; 5 C ) !8 8* >+ 0? ( 5 B+ - 153 & +8 8* 0? ; 5 C - "5 8* 5 &$) & ,1H B; 5 4@+ ? - ? - ,1H B > 8* 0? ; ? 5 4+ # B ? ; - 5 0 > 8* - 0? % 8 "C Dubious ORFs 81 0 5 '*, C - ( - G 8* 0 5 '*, 4+ - ( - G ; - - #$78* C 8$1 # -- 5 '*, BC 8$* - & 5 '*, K+ & ; 0 / - &6 - 0 - &+& 81 0 & 5 '*, C - ( - G ; - - &!* 781 + 8* 0 8 '*, < C - ( - G ; - - +'2 8'*< # 5 '*, "C + 8$* 5 +#; 5 "C ; 5 5 ; &.&1!E ( 2 81 G 8 4C &. ( - ( -- ( ( 81 $ + ? $ 5 "+ ; G G *5 !! *' ; ( 8'* & ) ( - & 5 @K+ - < *5 7 ( 8/1 & 1 *5J 5&>5 ; 5 C 4+ 5'175' 7& 4+ - *5 *5 ; 5 154 155 References Abeliovich, A., Schmitz, Y., Fariñas, I., Choi-Lundberg, D., Ho, W. H., Castillo, P. E., Shinsky, N., Verdugo, J. M., Armanini, M., Ryan, A., Hynes, M., Phillips, H., Sulzer, D., and Rosenthal, A., 2000. Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system. Neuron 25, 239-252. Ackerman, S. H., 2002. Atp11p and Atp12p are chaperones for F(1)-ATPase biogenesis in mitochondria. Biochim Biophys Acta 1555, 101-105. 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