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Differential Impact of Multiple Parkinson’s Disease Associated Genes on the Toxicity of α-Synuclein in a Yeast Model

Paul Jones
Lake Forest College
Lake Forest, Illinois 60045

 

LAKE FOREST COLLEGE

 

Senior Thesis

 

Differential Impact of Multiple Parkinson’s Disease Associated 

Genes on the Toxicity of α-Synuclein in a Yeast Model

 

by

 

Paul A. Jones

 

November 29, 2017 

 

The report of the investigation undertaken as a

Senior Thesis, to carry two courses of credit in

the Neuroscience Program

 

_______________________

Michael T. Orr

Krebs Provost and Dean of the Faculty

_______________________

Shubhik K. DebBurman, Chairperson

 

_______________________

Michael Kash

 

_______________________
Jean-Marie Maddux

 

ABSTRACT

 

Parkinson’s disease (PD) is characterized by α-synuclein misfolding and the death of midbrain neurons. PD cases are either familial (caused directly by a genetic mutation), or sporadic (influenced by multiple factors, including risk genes). One disease-causing gene is the α-synuclein protein, which has six PD-associated missense mutations. In budding yeast models, wild-type α-synuclein and the E46K, A53T, H50Q, and A53E mutants are cytotoxic and show varying degrees of membrane binding and aggregation, while A30P and G51D are non-toxic and show cytoplasmic diffusion. To explore the hypothesis that α-synuclein interacts with other loss-of-function PD genes to create toxicity, wild-type and mutant forms of α-synuclein were assessed in yeast with single deletions of PD-linked genes. Results indicate that certain PD-linked genes can moderate α-synuclein toxicity in broad or familial mutant-specific ways. Overall, this suggests that α-synuclein’s toxicity is mediated through multiple mechanisms, but each familial mutant operates through a specific mechanism of toxicity. 

 

DEDICATION



To my Grandpa, Don, 

and everyone affected by Parkinson’s disease and their families

 

ACKNOWLEDGMENTS

 

First, I would like to thank Dr. Shubhik DebBurman for giving me the opportunity to research in his lab and for his constant mentorship and guidance.  I have grown to appreciate the more arduous parts of the research process from my three years in his lab and I now know that my passion is attempting to understand the fundamental mechanisms of diseases and utilizing this understanding to better treat diseases. Dr. D has given me the tools to think like a researcher and helped shape my future career in biomedical research.

Next, I would like to thank my various mentors who have helped me cultivate lab skills especially Saul Bello Rojas ’16, Charles Alvarado ’16, and Emily Ong ’17. When I first started in lab I felt almost out of place and wasn’t sure what was going on, but now I feel at home in the lab and actually enjoy spending time running assays and reading scientific papers. I would also like to thank Beth Herbert for helping me develop proper lab practices and for being available whenever I had questions on lab procedures or anything else in Johnson.

 I would also like to thank the three students in lab whom I have mentored – Danielle Sychowski ’19, Ariane Balaram ’20, and Alexsandra Biel ’20 – who have contributed to my thesis or former projects.  Danielle was with me during the summer of 2016 when nothing seemed to be going right, but she helped me find humor in failure and helped troubleshoot problems that would later arise in my thesis work. Ariane and Alex were with me during the summer of 2017 and helped me collect so much data in such little time. Many of the experiments presented in chapter three of this thesis were done in coordination with them and it would have been impossible to complete my thesis without them.

 I would also like to thank my fellow thesis student, Rosemary Thomas ’18, for her constant support and presence in lab over the last three years, especially during this last chaotic and stressful semester. Additionally, I’d like to thank my other lab mates – Yoan Ganev ’19, Chisomo Mwale ’19, Joe Mountain ’20, and Niam Abeysiriwardena ’20 – for creating an engaging and honestly fun lab environment that made me excited to come in every day. I have to also thank Omid Saleh ’15, Emma Levine ’18 and Sam Gascoigne ’20 from Dr. Shingleton’s Lab, for their advice and support, especially during the late nights when I was at my most stressed and didn’t think I could make it through this process. I would like to thank my friends across campus who understand my passion for research and have been understanding of my reduced presence this last semester – especially those in Cross Country and in Delta Chi.

I don’t think it is possible to thank my family enough for all their support and for everything they have done for me, especially my mom and dad. They were always more than happy to drive four hours just to have lunch with me and have given everything they have to put me in the spot I am today. I love you both dearly. 

I would like to also thank the National Honor Society for neuroscience, Nu Rho Psi, for their financial support of my earlier project investigating α-synuclein’s interaction with BMAA and L-serine. Even though I was unable to complete this project as intended, I still feel honored to be one of four students selected to receive this grant in 2016.

Lastly, I would like to thank my committee members: Dr. Jean-Marie Maddux and Dr. Michael Kash, for helping me through this process and for their unique insight into my project.

 

 

TABLE OF CONTENTS

 

Abstract………………………………………………………………………………………………………………..i

Dedication…………………………………………………………………………………………………………….ii

Acknowledgements ……………………………………………………………………………………………..iii

List of Figures………………………………………………………………………………………………………vi

List of Tables………………………………………………………………………………………………………vii

List of Abbreviations…………………………………………………………………………………………..viii

Introduction ………………………………………………………………………………………………………….1

Methods and Materials …………………………………………………………………………………………32

Chapter 1: Characterization of α-Synuclein in BY4741, Δvps28, and across PD-Linked Gene Deletion Strains………………………………………………………………………………38

Chapter 2: Characterization of α-Synuclein in Δhsp31, Δatp13, and Δvps35…………………..57

Chapter 3: Characterization of α-Synuclein in Δvps13, Δsac1, and Δswa2……………………..78

Discussion ……………………………………………………………………………………………………………92

Conclusions ………………………………………………………………………………………………………….101

References ………………………………………………………………………………………………………….103

 

LIST OF FIGURES

 

Figure 1. α-Synuclein and PD pathology………………………………………………………………….5

Figure 2. α-Synuclein’s Cellular Function and Toxicity…………………………………………..10

Figure 3. Healthy Function of PD-linked Genes……………………………………………………….16

Figure 4. Toxic Mutations of PD-linked Genes………………………………………………………..17

Figure 5. Project Design……………………………………………………………………………….29

Figure 6. Predicted Phenotypes in yeast with and without PD-linked Gene Deletions…..30 

Figure 7. Localization of α-Synuclein in BY4741…………………………………………………….46

Figure 8. Toxicity and Protein Expression of α-Synuclein in BY4741…………………..50

Figure 9. Localization of α-Synuclein in Δvps28………………………………………………………51

Figure 10. Toxicity and Protein Expression of α-Synuclein in Δvps28…………………..53

Figure 11. Localization of WT α-Synuclein between Strains ……………………………54 Figure 12. Toxicity and Protein Expression of WT α-Synuclein between Strains…………56

Figure 13. Localization of α-Synuclein in Δhsp31…………………………………….….63 Figure 14. Toxicity and Protein Expression of α-Synuclein in Δhsp31…….…………….67

Figure 15. Localization of α-Synuclein in Δatp13.……………………………………….68 Figure 16. Toxicity and Protein Expression of α-Synuclein in Δatp13…….…………….72

Figure 17. Localization of α-Synuclein in Δvps35.……………………………………….73 Figure 18. Toxicity and Protein Expression of α-Synuclein in Δvps35…….…………….77 Figure 19. Localization of α-Synuclein in Δvps13.……………………………………….83 Figure 20. Toxicity and Protein Expression of α-Synuclein in Δvps13…….…………….85

Figure 21. Localization of α-Synuclein in Δsac1………………………………………….86 Figure 22. Toxicity and Protein Expression of α-Synuclein in Δsac1..…….…………….88 Figure 23. Localization of α-Synuclein in Δswa2.…….…………………………………..….89 

Figure 24. Toxicity and Protein Expression of α-Synuclein in Δswa2.…….…………….91

 

LIST OF TABLES

Table 1. α-Synuclein Constructs and Yeast Strains Used………………………………….37

 

ABBREVIATIONS

 

A30P: Familial PD point mutation, Alanine → Proline at the 30th amino acid

A53E: Familial PD point mutation, Alanine → Glutamic acid at the 53rd amino acid

A53T: Familial PD point mutation, Alanine → Threonine at the 53rd amino acid

AD: Alzheimer’s disease 

BY4741: Parent yeast strain without gene deletions

DA: Dopamine

E46K: Familial PD point mutation, Glutamic acid → Lysine at the 46th amino acid

eGFP: Enhanced green fluorescent protein

G51D: Familial PD point mutation, Glycine → Aspartic acid at the 51st amino acid

GFP: Green fluorescent protein

H50Q: Familial PD point mutation, Histidine → Glutamine at the 50th amino acid

HD: Huntington’s disease

MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxin

PD: Parkinson’s disease

PGK: Phosphoglycerokinase antibody

SC-Ura: Synthetic complete supplement mixture minus uracil

SNpc: Substantia nigra pars compacta

WT: Wild-type α-synuclein 

Δatp13: Yeast Knockout strain for probable cation-transporter 13 

Δhsp31: Yeast Knockout strain for heat shock protein 31 

Δsac1: Yeast Knockout strain for phosphatidylinositol-3-phosphatase 

Δswa2: Yeast Knockout strain for auxilin-like clathrin uncoating factor

Δvps13: Yeast Knockout strain for vacuolar protein sorting-associated protein 13 

Δvps28: Yeast Knockout strain for vacuolar protein sorting-associated protein 28 

Δvps35: Yeast Knockout strain for vacuolar protein sorting-associated protein 35

 

INTRODUCTION

Disease Arises from Genetic and Environmental Interactions

From the smallest bacteria to the largest blue whale, life’s diversity is astounding. Despite such amazing variation, all life is fundamentally composed of the same four macromolecules: lipids, sugars, proteins, and nucleic acids. In every organism, DNA is transcribed into RNA, which is later translated into proteins that provide a specific cellular function. Even the smallest change in DNA can radically alter the structure of the protein and its corresponding function, potentially leading to disease. In a few diseases, a single genetic mutation results in the pathogenesis of the disease. For example, mutations in the gene huntingtin cause nearly all cases of Huntington’s disease (HD). Healthy individuals have a huntingtin gene with less than thirty-seven CAG nucleotide base-pair repeats but individuals with HD have more than of thirty-seven CAG repeats (Gusella et al., 1983). This expanded CAG region increases the amount of glutamine amino acids in the final protein, which causes aggregation in the nucleus of the cell, ultimately leading to disease (Rubinsztein and Carmichael, 2003). However, most diseases are not so straightforward and result from a complicated interaction between multiple genes and various environmental factors. Schizophrenia has hundreds of identified genetic and environmental factors that increase the risk of developing the disease, but no single genetic or environmental factor can cause the disease (Ripke et al., 2014; McDonald and Murray, 2000). The bulk of neurological diseases like Alzheimer’s disease (AD) and Parkinson’s disease (PD) appear to behave in this way. Given how most diseases are influenced by both genes and the environment, it is essential to understand how both genes interact with environmental factors and how genes interact with other genes.

Neurodegenerative Diseases

The structure of the nervous system makes it particularly vulnerable to disease in ways that other systems are not. First, the entire nervous system is extensively interconnected and even slight amounts of structural damage can disrupt this complex network and alter behavior. This sensitivity is compounded by the fact that the nervous system only replenishes a negligible number of new neurons as an adult (Eriksson et al., 1998) and actively produces chemicals that inhibit the growth and development of new neurons (Chen et al., 2000). Simply put, the brain is too complicated to replace individual neurons and have their function remain intact. In fact, alterations in neuronal connectivity contribute to diseases like schizophrenia, ADHD, autism (Di Martno et al., 2014) and, after injury, promote the development of chronic pain (Woolf and Doubell, 1994). Second, the blood-brain barrier isolates the brain from the rest of the body, which protects it from certain toxins and infections, but also impairs the clearance of cellular debris and prevents the immune response from acting against pathogens in the brain.

These points are both particularly relevant to neurodegenerative diseases, a class of diseases defined by the progressive loss of nervous tissue and corresponding loss of bodily function. These diseases will continue to become more impactful to society as humans continue to live longer. In the most common neurodegenerative disease, Alzheimer’s disease (AD), cells in the hippocampus and cortex that are responsible for memory formation and storage gradually die, resulting in cognitive dysfunction and memory loss. Slowly, more and more neurons in other brain systems die, resulting in an increased loss of function. Eventually, neurons in the brainstem that are responsible for swallowing die, leading to the patient’s death (Förstl and Kurz, 1999). In the case of Huntington’s disease (HD), cells in the striatum that are responsible for movement regulation die, and motor control is lost. 

Upon closer inspection, another key similarity is present among nearly all neurodegenerative diseases. If the surviving neurons of a patient are observed, a substantial number would show large aggregates of various misfolded proteins. In AD, both amyloid-β plaques and neurofibrillary tau tangles form throughout the cortex and hippocampus, contributing to the loss-of-function of these regions (Hardy and Selkoe, 2002). In HD, aggregates of Huntingtin protein develop in the striatum, and in Parkinson’s disease (PD) aggregates of α-synuclein called Lewy Bodies appear in the midbrain. This thesis will focus on α-synuclein and its role in PD.

 

Parkinson’s Disease

PD is the second most common neurodegenerative disease with a prevalence rate between 0.3 % and 1.0 % for those over sixty, affecting an estimated six million people worldwide (De Lau and Breteler, 2006; Zhang and Román, 1993). PD has an average age of onset of about sixty, but risk increases with age (Nass and Przedborski, 2008). Additionally, many cases of PD occur much earlier. PD is characterized by six hallmark motor symptoms, with three occurring earlier in the progression of the disease– a resting tremor, bradykinesia, and muscle rigidity – and three occurring later – freezing of gait, flexed posture, and loss of postural reflexes. Most patients also experience non-motor symptoms such as mood disorders, insomnia, cognitive impairment, and sensory loss, particularly in olfaction (Park and Stacy, 2009). As the disease progresses, patients continually lose motor function, eventually becoming wheelchair-bound and bedridden. While PD is generally not fatal, it massively increases the risk of death due to aspiration pneumonia or complications due to falls from motor impairment (Poewe, 2006). 

The brain of a PD patient looks very similar to that of a healthy individual at first glance, but one small brain region is majorly altered. Most of PD’s symptomology can be traced back to the substantia nigra pars compacta (SNpc), an extremely small, highly specialized region of the brain about 1-2 mm in size (Olanow and Tatton, 1999). Normally, the SNpc produces dopamine and projects to the caudate and putamen of the striatum, which are the brain regions responsible for regulating the timing and coordination of voluntary muscle movements. When observed with the naked eye, this region is normally noticeably darker than the rest of the brain, due to the unique presence of the pigment neuromelanin (D’Amato et al., 1986). However, if the brain of an individual with PD is observed, this dark band of cells is no longer present. In fact, an estimated 50-70% of the dopamine producing neurons in the SNpc have already died and an 80% reduction of dopamine in the striatum is present at the onset of PD’s symptoms (Ross et al., 2004; Dauer and Przedborski, 2003). The brain pathology of PD is depicted in Figure 1.  This reduction in dopamine release due to SNpc degeneration disrupts levels of excitation and inhibition to downstream regions in the striatum and basal ganglia. This disruption results in slowed and unintended movements, corresponding with PD’s symptoms of bradykinesia and a resting tremor (Halliday et al., 2014). 

Most current treatments for PD focus on restoring normal levels of dopamine to the striatum. The most common and effective treatment is L-DOPA supplementation, which utilizes a precursor of dopamine to normalize levels of dopamine produced and released by the SNpc (Marsden and Parkes, 1977). Other less common pharmacological treatments utilize dopamine receptor agonists to mimic the effect of dopamine, or drugs which inhibit the enzymes that normally degrade dopamine (Schapira et al., 2006). These 

 


Figure 1. α-Synuclein and PD pathology

 

In healthy individuals, a thick dark band of dopaminergic neurons is present in the substantia nigra, and α-synuclein remains soluble. In individuals with PD, there is a substantial atrophy in the substantia nigra. Upon closer inspection, many dopaminergic neurons in this region have died and others produce less dopamine. This region is also filled with oligomerized and aggregated α-synuclein, which underlies PD’s pathology.




treatments are very effective, but lose efficacy as the SNpc continues to degenerate and come with a host of side effects. Common minor side effects include insomnia, headaches and dry mouth, but severe side effects such as psychosis and cardiac events are not uncommon (Schapira et al., 2006). An even more aggressive form of PD treatment exists in the form of deep-brain stimulation, which uses electrodes to restore the normal activity of the brain regions targeted by SNpc neurons (Deuschl et al., 2006). Given that the current treatments for PD have a limited efficacy window and have potential severe side effects, it is necessary to research and develop new treatments for PD.

 

Sporadic and Familial PD

PD can broadly be divided into two classes, sporadic, without explicit cause, or familial, which can be traced to a specific genetic mutation. Sporadic PD is the most common form of the disease and is responsible for 85-90% of total cases (Schiesling et al., 2008). Sporadic PD generally occurs later in life (relative to familial PD) and has an average age-of-onset of sixty years of age. Multiple non-genetic factors moderate the probability of developing PD. Pesticide exposure, heavy metal toxicity, and methamphetamine use have been linked to an increased risk while tobacco use, alcohol consumption, and coffee consumption have been linked to a reduced risk (Dick et al., 2007; Kalia and Lang, 2015; Popat et al., 2013). Recent genome-wide association studies have also identified many risk genes and loci that increase the probability of developing PD without necessarily causing the disease (Lill et al., 2012). Some of the more well established risk genes and loci include MAPT, (Edwards et al., 2010) PARK16, (Simon-Sanchez et al., 2009), and VPS13C (Nalls et al., 2014). 

Familial PD differs from sporadic PD in that it has an explicit genetic origin. While sporadic PD rarely occurs in individuals under sixty, familial PD regularly occurs in the forties and fifties and sometimes as early as the twenties (Schrag et al., 1998). Despite being responsible for only 10% to 15% of cases of PD, familial PD has been extensively studied and many disease-causing genes have been identified and thoroughly studied. Autosomal dominant, gain-of-toxicity PD genes include SNCA (Polymeropoulos et al., 1997) and LRRK2 (Funayama et al., 2002). Autosomal recessive, loss-of-heathy-function PD genes include Parkin (Lücking et al., 2000), PINK1(Valente et al., 2004), ATP13A2 (Al-Din et al., 1994), and DJ-1 (Bonifati et al., 2003). Regardless of their specific cause, both familial and sporadic PD have the same underlying pathology: the toxic aggregation of α-synuclein in the SNpc and the degeneration of dopaminergic neurons. Thus, α-synuclein must be a key component in the progression of PD. This thesis will focus on α-synuclein, which is coded by the SNCA gene, and α-synuclein’s interaction with autosomal recessive PD-causing and PD-risk genes.

 

α-Synuclein

α-Synuclein is a protein broadly expressed in the human brain and is normally found at the presynaptic terminal of neurons (Kahle, 2008). It was first identified in relation to PD when it was found to be the primary component of Lewy Bodies found in the SNpc of PD patients (Pollanen et al., 1993). Its normal biological role is still controversial but there is evidence to suggest it assists in vesicle trafficking through the formation of SNARE-complexes, which allow dopamine to be released and help vesicles form (Burré et al., 2010). Deletion of α-synuclein in rodents fails to yield any obvious phenotype changes while deletion of α-synuclein and β-synuclein, a similar protein in function and structure, reduces dopamine release by 20% (Chandra et al., 2004). In rodents with α-, β-, and γ-synuclein deleted, widespread alterations in neuronal firing and shortened lifespans are present, but no specific change occurs in the SNpc (Greten-Harrison et al., 2010). Thus, it appears α-synuclein toxicity is due to a gain-of-toxic-function mechanism in PD.

α-Synuclein’s potential function and mode of toxicity is directly linked to its structure. α-Synuclein is a 140-amino acid long protein with a natively unfolded conformation, meaning that its structure is highly flexible, and it has multiple stable three-dimensional conformations (Uéda et al., 1993; Weinreb et al., 1996). It has three distinct domains, each with its own defined functions. The N-domain is composed of amino acids 1-60 and has an amphipathic helical structure, helping it bind membranes and vesicles (Ulmer et al., 2005). The M-domain is composed of amino acids 61-95 and contains a highly hydrophobic NAC (non-amyloid-β component) region, which is crucial to the oligomerization and aggregation of α-synuclein (Giasson et al., 2001; Bodles et al., 2001). Finally, the C-domain is composed of amino acids 96-140 and has many acidic residues. This region is particularly disordered and is thought to keep α-synuclein soluble (Li et al., 2005). In living systems, α-synuclein exists in multiple states: unstructured monomeric α-synuclein that is soluble in the cytosol (Kim, 1997), helical monomeric α-synuclein bound to cell membranes (Jao et al., 2004), stable, soluble complexes of multiple α-synuclein subunits called oligomers (Tsigelny et al, 2008), and large insoluble aggregates called Lewy Bodies that are found in PD (Pollanen et al., 1993). In healthy individuals, α-synuclein is largely found soluble in the cytosol or bound to the membrane, but in PD, most α-synuclein is found in larger aggregates and oligomers, as described in Figure 1.

The fundamental question that remains is: how does α-synuclein become toxic in PD? Initially, it was thought that Lewy Bodies themselves may be toxic, but it appears that they act to isolate or neutralize oligomeric α-synuclein (Braak et al., 2007; Auluck et al., 2010). There is growing evidence that oligomeric α-synuclein is the primary driver of α-synuclein toxicity. Oligomeric α-synuclein disrupts membrane stability and impairs intracellular transport (Auluck et al., 2010; Marques and Outeiro, 2012). Additionally, α-synuclein, when modified in a way that prevents oligomer formation, is non-toxic despite still forming aggregates and binding to membranes (Soper et al., 2008; Burré et al. 2015). However, Lewy Bodies may propagate α-synuclein aggregation throughout the brain by seeding oligomers in regions beyond the SNpc, explaining the more widespread neurodegeneration in later stages of PD (Volpicelli-Daley et al., 2011). Monomeric α-synuclein appears to be non-toxic itself but assists in forming larger oligomeric species when at high concentrations or under certain conditions (Wright et al., 2009). A few α-synuclein modifications that are thought to modulate its toxicity include phosphorylation, which alters α-synuclein’s localization and interaction with other proteins (Fiske et al., 2011a; Oueslati, 2016), oxidative stress, which promotes and stabilizes oligomers (Norris et al., 2003), lysine modifications like SUMOylation, which prevent oligomerization (Oh et al., 2011), and glycation, which promotes oligomerization and increases oxidative stress (Shaikh and Nicholson, 2008). Figure 2 summarizes the normal function of α-synuclein and how α-synuclein becomes toxic in PD. In summary, multiple factors contribute to how α-synuclein oligomerizes and becomes toxic in PD. 

 

Figure 2. α-Synuclein’s Cellular Function and Toxicity

In healthy individuals, α-synuclein is usually found at the synapse on the membrane and assists in the release of dopamine (1). It is also degraded by the lysosome (2). In PD α-synuclein oligomerizes and aggregates, which impairs normal dopamine release (3), impairs endocytosis (4), disrupts membrane stability (5), and impairs lysosomal degradation. It is also thought to increase oxidative stress (6), which promotes DNA damage, damages mitochondria, and in turn increases α-synuclein aggregation.

 

Thus, it makes sense that multiple mutations in the SNCA gene, which codes for α-synuclein, cause early-onset PD.   

 

 Familial α-Synuclein Mutations

Two types of SNCA mutations exist that cause familial PD, multiplications and point mutations. Multiplication mutants are either duplications or triplications of the SNCA gene, which result in double or triple the amount of α-synuclein expression. These mutations cause autosomal-dominant PD that has an average age of onset of 48.4 and 34 years of age, respectively (Chartier-Harlin et al., 2004; Singleton et al., 2003).  These mutations show that toxicity can result from increased levels of α-synuclein expression, while still showing similar pathology to sporadic PD. The other set of mutations is point mutations, which occur due to a single amino acid in α-synuclein being converted into another. For example, an alanine in the fifty-third position is converted into threonine in the A53T mutation, the first identified α-synuclein point mutation. The A53T mutation and two other mutations, A30P and E46K, were identified in the late 1990s and early 2000s and have extensive bodies of associated published research. The other three point mutations, G51D, H50Q, and A53E, were discovered in the early 2010s and have comparatively tiny bodies of research. This thesis will explore the interactions that all six point mutations have with other PD-linked genes.

 

A30P, E46K, and A53T

What would later be identified as the A53T mutant was first described in 1990 in two kindreds in southern Italy (Golbe et al., 1990). This mutation was also later identified in three other kindreds (Polymeropoulos et al., 1997). The mean age of onset for these individuals was forty-six and their symptoms were similar to sporadic PD but with less incidence of tremors (Spira et al., 2001). When compared to wild-type α-synuclein (WT), α-synuclein without any genetic mutations, A53T α-synuclein is disordered and soluble at low concentrations but rapidly aggregates at higher concentrations into fibrils (Conway et al., 1998). Cell culture also shows that A53T α-synuclein oligomerizes faster than WT α-synuclein (Sharon et al., 2015). A53T transgenic rodent models show broad neurodegeneration, α-synuclein membrane binding, and motor impairment but no specificity to the SNpc nor dopaminergic loss (Lee et al., 2002; Jensen et al., 1998; Crabtree and Zhang, 2013). In budding yeast models, A53T binds to membranes like WT (Sharma et al., 2006), but is toxic and accumulates in fission yeast (Brandis et al., 2006). 

The A30P mutation was first identified in a British family in 1998 (Krüger et al., 1998). A30P α-synuclein is disordered and soluble at low concentrations but forms amorphous spheres rather than fibrils like A53T at high concentrations (Conway el al., 1998). Unlike WT and A53T, A30P is unable to bind to membranes and remains cytoplasmic diffuse (Jensen et al., 1998). This altered phenotype is likely due to a disruption in the N-domain α-helix structure, which assist in membrane binding, by adding a disruptive proline to the middle of the α-helix structure (McLean et al., 2000). This diffuse phenotype corresponds with findings both budding and fission yeast (Sharma et al., 2006; Brandis et al., 2006). Rodent models transfected with A30P α-synuclein do show SNpc degradation, Lewy-body-like pathology, and substantial dopaminergic loss, but remain without motor impairment (Klein et al., 2002). A30P also forms stable oligomers, which have distinct appearances compared to WT and A53T (Conway et al., 2000a). 

The E46K mutant was first described in a Spanish family with a variant of PD called dementia with Lewy Bodies, which has both PD and dementia symptoms (Zarranz et al., 2004). E46K α-synuclein quickly forms large fibrils at a similar rate to A53T but shows less oligomerization (Fredenburg et al., 2007; Conway et al., 2000b). E46K also binds more tightly to membranes than WT and induces high toxicity in mammalian cell cultures, despite no changes in aggregation (Bodner et al., 2010; Íñigo-Marco et al., 2017). E46K’s toxicity does not appear to be caused by increased phosphorylation levels (Íñigo-Marco et al., 2017; Fiske et al., 2011b). In budding yeast, E46K binds to lipid membranes and remains relatively non-toxic. In fission yeast, E46K accumulates on the endomembrane system and becomes toxic (Fiske et al., 2011b), potentially through impairing protein trafficking. Despite much research, the mechanism of E46K toxicity remains elusive.

 

G51D, H50Q, and A53E

After about ten years with no new mutants identified, H50Q was identified as a distinct mutation first in an English patient, then in a Canadian patient, both in 2013 (Proukakis et al., 2013; Appel-Cresswell et al., 2013). This mutation results in PD with dementia symptoms, with both patients presenting symptoms at about sixty years of age. H50Q α-synuclein appears to have no major structural alterations and oligomerizes at a rate similar to WT but rapidly aggregates (Ghosh et al., 2013). H50Q also induced mitochondrial fragmentation and toxicity in cell cultures, but the underlying mechanism is unclear (Khalaf et al., 2014). Interestingly, the H50 site is a key copper-binding site for α-synuclein and the H50Q mutant in mammalian cell cultures with heightened level of copper levels shows increased toxicity and altered aggregation compared to WT under the same conditions (Villar-Piqué et al., 2017). Cell cultures expressing sH50Q are also particularly sensitive to oxidative stress (Xiang et al., 2015). In budding yeast, H50Q α-synuclein has similar localization and toxicity to WT, but it binds to the endomembrane system and is somewhat less toxic in fission yeast (Tembo, Thesis, 2015). The cause of toxicity for H50Q is still unclear, but multiple mechanisms of toxicity may be present.

The G51D mutant was identified in a British family in 2013 (Lesage et al., 2013a). This mutation expressed itself in a particularly aggressive form of PD with an age of onset in the mid-thirties and death within five to seven years (Kiely et al., 2013). In addition to PD-symptoms, patients also presented with pyramidal symptoms, psychiatric issues, spasticity, and autonomic dysfunction. The brains of these patients contained SNpc degeneration along with lesioning to the striatum with prominent α-synuclein aggregation in both regions. Neuronal cell culture models show that G51D α-synuclein fails to bind to membranes and slowly aggregates in amorphous aggregates or remains cytoplasmically diffuse (Fares et al., 2014; Rutherford et al, 2014). G51D is also hyper-phosphorylated, results fragmented mitochondria, and localizes to the nucleus. G51D behaves similar to A30P in budding yeast – cytoplasmic diffusion and relatively non-toxic – and in fission yeast – less aggregation and relatively non-toxic (Tembo, Thesis, 2015).

The A53E mutation is the most recently identified mutant, being identified in a Finnish family in 2014 (Pasanen et al., 2014). The first identified patient was thirty-six at the time of diagnosis and presented with PD and pyramidal symptoms, spasticity, and insomnia. She died at the age of sixty. The brain and spinal cord of this patient showed broad neurodegeneration but particular dopaminergic loss in the SNpc. The little research done on A53E α-synuclein indicates that it results in reduced aggregation and membrane binding and is cytotoxic (Lázaro et al., 2016; Ghosh et al., 2014; Rutherford and Giasson, 2015). However, whether oligomerization is involved is inconclusive with Lázaro et al. indicating it is reduced, Ghosh et al. showing no effect, and Rutherford and Giasson showing that it is increased. Further analysis has shown mitochondrial and Golgi fragmentation with A53E. In budding yeast, A53E binds less to the membrane and has about the same toxicity as WT, while in fission yeast, A53E has reduced aggregation (Tembo, Thesis, 2015).

 

Other PD-linked Genes

Studying the role of α-synuclein mutations alone will help assist in understanding α-synuclein’s mode of toxicity and how PD occurs. However, mutations on α-synuclein account for less than 1% of total cases of PD. PD is not a monogenetic disease and many other well-established genes contribute to both familial and sporadic PD. A large majority of these genes regulate the same cellular functions that are affected in PD, most notably protein trafficking, oxidative stress management, vesicle formation, and metal toxicity. Many of these mutations have evidence to support a loss-of-healthy-function mechanism. The normal function of a few of these PD-linked genes is described in Figure 3, while the effects of the mutations is shown in Figure 4.  This thesis will focus on the interaction between WT and familial mutant α-synuclein and other PD-linked genes. The following section describes the human and yeast genes analyzed in the study. 

 

Figure 3. Healthy Function of PD-linked Genes

Multiple genes are involved in PD and are thought to function through pathways linked to α-synuclein toxicity. VPS35 assists in the transport of various proteins, including α-synuclein, to either the lysosome, Golgi, or membrane (1). PINK1 and Parkin function together to stabilize mitochondria and reduce oxidative stress (2). DJ-1 reduces oxidative stress, stabilizes mitochondria, and directly and indirectly assists in protein degradation through transcription activation (3). VPS13C stabilizes the mitochondria and prevents mitochondrial destruction (4). ATP13A2 pumps metal ions like manganese out of the cell and prevents metal toxicity (5). DNAJC6 assists in vesicle formation (6) and SYNJ1 removes coating protein to form mature vesicles (7).

 

Figure 4. Toxic Mutations of PD-linked Genes

If a certain PD-linked gene is mutated, a specific protective cellular function is lost. These mutations often compound with each other and promote α-synuclein toxicity.  VPS35 mutations prevent normal protein transport and degradation and cause α-synuclein to accumulate on vesicles (1). PINK1 and Parkin mutations damage mitochondria and promote oxidative stress, which causes DNA damage and increase α-synuclein accumulation (2). DJ-1 mutations also increase oxidative stress, destabilize mitochondria, and promote α-synuclein accumulation (3). VPS13C mutations promote mitochondria degradation (4). ATP13A2 mutations prevent the clearance of toxic metals (5). DNAJC6 mutations prevent full vesicle formation (6) and SYNJ1 mutations prevent vesicle decoating (7), both which impair normal neurotransmission.

 

Disease-Causing Genes

DJ-1

DJ-1, coded by PARK7, is a small, 189 amino acid long protein broadly expressed throughout the body (Honbou et al., 2003). It has multiple proposed functions from regulating male fertility, transcriptional regulation, and being an oncogene product, but the function relevant to PD is that of a hydroperoxide-response chaperone protein (Ariga et al., 2013). DJ-1 has multiple functions thought to reduce oxidative damage. These include moving the transcription factor Nrf2 to the nucleus to activate multiple antioxidant genes (Clements et al, 2006), protecting mitochondria against oxidative degradation (Junn et al., 2009), and acting as a protease that assists in the degradation of aggregated proteins (Koide-Yoshida et al., 2007). Multiple PD-linked DJ-1 mutations exist including single amino acid deletions, additions, substitutions, and large domain deletions (Kahle et al., 2009). These mutations alter DJ-1’s structure and reduce or completely eliminate DJ-1’s normal function. Without DJ-1, oxidative stress builds up in the neurons of the SNpc and causes widespread damage to the cell, particularly by promoting α-synuclein oligomerization (Norris et al., 2003). Gradual loss of DJ-1 function is linked to sporadic PD, even without mutations (Bandopadhyay et al., 2004). Yeast Hsp31 is a highly-conserved protein similar in structure and functions to DJ-1. Like human DJ-1, it acts as a chaperone by reducing oxidative stress and stabilizing mitochondria (Aslam and Hazbun, 2016)




ATP13A2

ATP13A2, coded by PARK9, belongs to a large superfamily of ion pumps responsible for moving various ions of salt, metal, and hydrogen across the membranes of a cell (Schultheis et al. 2014). ATP13A2 is specifically responsible for pumping toxic Mn2+ and other heavy metal ions out of the cell. Long term-manganese exposure is an established risk factor for PD and heightened levels of manganese and iron are present in the brains of patients with PD (Gorell et al., 1997; Hirsch et al. 1991). Excessive levels of heavy metals can radically disrupt many cellular processes, particularly in sensitive regions like the brain where metals can alter neurotransmitter receptor activity. Multiple PD-linked ATP13A2 mutations exist in the population, all of which impair metal transport by reducing or eliminating ATP13A2’s normal function (Yang and Yanming, 2014). One ATP13A2-linked form of PD is Kufor-Rakeb syndrome, which presents in adolescence with PD symptoms, pyramidal symptoms, spasticity with corresponding neurodegeneration due to excesses iron accumulation in the brain (Schneider et al., 2010). ATP13A2 mutations also enhance the oligomerization and accumulation of α-synuclein in cell culture models (Lopes da Fonseca et al., 2016). The yeast orthologue of human ATP13A2 is Ypk9p, which reduces toxicity in yeast in response to multiple metals including Cd2+, Mn2+ and Ni2+ (Schmidt et al., 2009).

 

VPS35

VPS35 belongs to a broad class of highly conserved proteins called vacuolar protein sorters. VPS proteins are involved in transporting proteins and other materials in endosomes to their destinations in the cell. VPS35 is one of the primary components of the retromer complex, which is a large group of proteins that assists in the retrograde transport from small endosomes to either the Golgi or to the cell membrane (Seaman, 2012). While multiple PD causing mutations exist in VPS35, the mutation which has the most cases and largest body of associated research is D620N (Deng et al., 2013). This mutation follows an autosomal dominant pattern of inheritance, but it does not appear that it’s toxicity is due to a gain-of-function. The mutation does not affect the structure of the retromer complex but causes aberrations in receptor transport and selective SNpc degradation (Follett et al, 2014; Tang et al., 2015). Drosophila models have indicated that VPS35 activity is essential for proper α-synuclein degradation through the lysosome (Miura, et al., 2014). Thus, loss of VPS35 should increase α-synuclein accumulation and toxicity. In yeast, VPS35 appears to have an identical role to that present in higher level organisms. The loss of VPS35 is not toxic to yeast itself. However, it does become toxic when another protein, EIF4G1, is overexpressed (Dhungal et al., 2015).  

 

VPS13C

Like VPS35, VPS13C is a vacuolar protein sorter. Very little is known about VPS13C but it has established activity in glucose metabolism and insulin regulation (Windholz et al., 2013; Grarup et al., 2010). Genome-wide association studies have been inconclusive in demonstrating that VPS13C is a definite risk factor for PD (Safaralizadeh et al., 2016; Chen et al., 2016). However, certain VPS13C mutations have been shown to cause PD (Lesage et al., 2013b), but the number of cases is limited. The loss of normal VPS13C function promotes mitochondrial dysfunction and activates mitophagy, a process of mitochondrial self-destruction. The corresponding yeast orthologue is VPS13, which also binds mitochondria and prevents mitophagy (Park et al., 2016).

 

SYNJ1 and Sac1

SYNJ1 codes for synaptojanin, a protein highly expressed in neuronal tissue, particularly in the presynaptic cleft (Cremona et al., 1999). It assists in neurotransmission  by forming new vesicles and endosomes from the membrane, which can be filled with neurotransmitters. SYNJ1 has a phosphatase activity in a small Sac1 domain, which removes phosphates from other proteins involved in endocytosis (McPherson et al., 1996). The removal of these phosphates results in the formation of functional vesicles from protein-coated intermediates. PD mutations linked to SYNJ1 were first identified in 2013 and cause a loss-of-function in the Sac1 phosphatase domain (Krebs et al., 2013; Olgiati et al., 2014). These patients presented with an early-onset, autosomal recessive PD with typical symptoms, cognitive impairment, and seizures. The Sac1 domain is functionally conserved in yeast – it maintains its phosphatase activity and assists in vesicle trafficking (Guo et al., 1999). 

 

DNAJC6 and Swa2

DNAJC6 codes for the protein auxilin, a small catalytic heat shock protein (Ohtsuka et al., 2000). Like SYNJ1, DNAJC6 assists in the formation of endosomes and vesicles by cleaving partially formed-vesicular buds into protein-coated vesicles (Park et al., 2015). Multiple PD mutations exist in the DNAJC6 gene, which follow an autosomal recessive pattern (Olgiati et al., 2016). Patients present with a juvenile form of PD, severe cognitive impairment, and epilepsy (Köroğlu et al., 2013). It is hypothesized that DNAJC6 mutations result in the accumulation of partially formed vesicles at the presynaptic cleft and a massive reduction in neurotransmission (Krantz et al., 2013). The yeast orthologue of auxilin is Swa2, which maintains its role in the formation of complete protein coated vesicles (Krantz et al., 2013). Even though, SYNJ1, DNAJC6, and α-synuclein all assist in vesicle formation, virtually no research has been done on their interactions in model systems of PD.

 

Gap in Knowledge

Despite the large body of research into PD’s genetics, much remains to be learned. While the three old familial mutants on α-synuclein (A30P, E46K, A53T) have extensive bodies of research, their definite mode of toxicity is still unclear, particularly for E46K. The new mutants (G51D, H50Q, A53E) have steadily increasing bodies of literature but still have no well-established modes of toxicity. The H50Q mutant has many supported means of toxicity, but the ultimate cause is still controversial. Other genes involved in PD have variable levels of research done, but they generally have more well-established cellular functions. DJ-1, ATP13A2, and VPS35 are responsible for oxidative stress management, metal transport, and retrograde transport, respectively. VPS13C, SYNJ1, and DNAJC6 are involved in endocytosis and vesicle formation. The bulk of research into these genes so far has looked at them in isolation without the effects of α-synuclein present. However, this is not representative of what occurs in PD patients. The manner in which each mutation functions in PD is due to a series of complex interactions with multiple proteins, especially α-synuclein. Additionally, α-synuclein’s familial mutants are likely to have compounding effects with other PD-genes if they share a similar mode of toxicity, but this has yet to be explored. This thesis will examine whether WT α-synuclein’s toxicity is exacerbated by the loss-of-function of various of PD genes in yeast. It will also explore whether the toxicity of each familial mutant is altered by the loss-of-function of PD-linked genes.

 

Yeast as a Model

Various models have been utilized in the study of PD including primates, rodents, Drosophila, C. elegans, and mammalian cell cultures. Primate models are the most genetically and physiologically similar to humans but come with a host of logistical and ethical issues. Most primates use a MPTP toxin model that fails to replicate PD’s slow progression and is non-toxic in some primate species (Nass and Przedborski, 2008). Rodent research is effective in displaying PD symptomology, but often uses the same toxin model as primates, with the same flaws (Nass and Przedborski, 2008). Additionally, α-synuclein rodent models, particularly those for the familial mutants, often fail to mimic the complete symptomology and pathology of PD (Lee et al., 2002; Crabtree and Zhang, 2013). Neural cell culture models are effective in displaying pathology but fail to capture the complicated connections present in the brain and often have a morphology distinct from human tissue (Nass and Prezedborski, 2008; Banker and Goslin, 1998).

While it may seem counterintuitive to study a complex neurological disorder using a single cell organism, yeast remains a powerful model organism for many diseases, particularly PD. Even though yeast is a simple organism it retains many of the same functional genes and proteins involved in fundamental cellular processes including those for protein trafficking, mitochondrial regulation, and stress management – the same processes affected by PD. 

Furthermore, multiple yeast models have already been established for many human neurodegenerative diseases including AD (Komano et al., 1998) and HD (Meriin et al., 2002). Various yeast models of PD of have been developed using both budding and fission yeast (Outeiro and Lindquist, 2003; Zabrocki et al., 2005; Willingham et al., 2003; Brandis et al., 2006). α-Synuclein is not native to yeast, but can be easily expressed through multiple methods, with most models using a plasmid expression vector. Outeiro and Lindquist have shown that α-synuclein is toxic to yeast in a dose-dependent manner with higher levels of α-synuclein impairing normal growth and increasing levels of aggregation. Overall, budding yeast models show α-synuclein toxicity, localization, aggregation, the same phenotypes seen in PD. Deletions to many yeast genes that are involved in the fundamental cellular process affected in PD – protein transport, mitochondrial health, membrane interaction, and stress management – increase α-synuclein’s toxicity to yeast (Willingham et al., 2003). Other gene deletions have been shown to affect the toxicity of certain familial mutants of α-synuclein while leaving WT or other mutants unaffected (Liang et al., 2008). A budding yeast model will be used throughout this thesis with a plasmid vector system expressing a moderate amount of α-synuclein. Budding yeast was chosen due to the availability of genetic tools that allow for gene deletions and for its ability to mimic the same fundamental α-synuclein phenotypes present in PD.



Hypothesis and Aims

Hypothesis and Project Overview

Overall, the purpose of this thesis was to better understand the mechanisms of toxicity behind WT and familial mutant α-synuclein and six other PD-linked genes in budding yeast. This was assessed by inserting WT and familial mutant α-synuclein into strains of yeast that mimic the same loss-of-function mutants seen in PD patients for six genes, then observing alterations in their phenotypes compared to yeast without and gene loss-of-functions mutations. None of the PD-linked genes deletions analyzed in this study had previously been shown to be toxic with WT α-synuclein, so it was hypothesized that they would not increase WT α-synuclein’s toxicity or alter other yeast phenotypes. It was hypothesized that certain gene deletions would increase α-synuclein’s toxicity and aggregation in a familial mutant specific manner so that only one or two mutants may become toxic in each strain. This would indicate that the familial mutant and loss-of-function PD gene potentially share a similar mechanism of toxicity, opening paths for future analysis of the exact nature of this interaction. An overall schematic view of this project is presented in Figure 5.

 

Phenotypes Assessed

The phenotypes assessed throughout this study were α-synuclein localization, toxicity, and protein expression levels. α-synuclein localization was assessed using GFP microscopy. GFP microscopy utilizes a fluorescent-tag molecule bound to α-synuclein to visualize its cellular location under a microscope and to determine if α-synuclein appears cytoplasmically diffuse, bound to the membrane, or in aggregates. Cell toxicity was assessed using a serial-dilution spotting assay. This assay compares α-synuclein’s effects on yeast growth across different yeast strains and with different mutants of α-synuclein. Differences in yeast growth indicate variations in α-synuclein toxicity. Protein levels were measured using western blotting analysis. Western analysis compares levels of α-synuclein present between different samples and can be used to determine if levels of toxicity or aggregation correspond with increased α-synuclein levels.

 

Chapter 1

The purpose of chapter one was to establish a baseline for comparison in future chapters and to examine the effects that the gene deletions had on WT α-synuclein. The first subsection of Chapter 1 replicated previously work done on WT and the six familial mutants in yeast with no gene deletions (BY4741) to ensure that phenotypes were consistent with previously established results. The next subsection of Chapter 1 looked at  WT and familial mutant α-synuclein in yeast with the vps28 gene deleted (Δvps28). Δvps28 is an established enhancer of WT α-synuclein toxicity and aggregation but the effects that it has on the familial mutants had not been thoroughly explored (Willingham et al., 2003; Senagolage, Thesis, 2012). It also served as a positive toxicity control for measuring levels of toxicity. Lastly, WT α-synuclein was assessed between BY4741, Δvps28, and the six PD-gene deletion yeast strains used in this study. This was done to determine if any gene deletions had broad effects on α-synuclein phenotypes.

 The expected phenotypes for BY4741 and hypothesized phenotypes for a specific familial interaction in any strain are presented in Figure 6. BY4741 phenotypes should match previously established results from Kulkulka (Thesis, 2013) and Tembo (Thesis, 2015), which they overwhelmingly did. WT, E46K, A53T, H50Q, and A53E showed relatively moderate and nearly equal amounts of toxicity, similar localization patterns, and nearly equal levels of protein expression. A30P and G51D showed a non-toxic phenotype, cytoplasmically diffuse localization, and slightly elevated α-synuclein levels. WT α-synuclein was slightly more toxic and showed increased aggregation in Δvps28, but the overall trend for the familial mutants in Δvps28 was similar to BY4741 and no particular familial mutant interaction was present. Three unexpected PD-linked gene deletion strains also increased WT α-synuclein’s toxicity and showed increased levels of aggregation.

 

Chapter 2

The purpose of chapter two was to assess the effects that Δhsp31, Δatp13, and Δvps35 had on the phenotypes of WT and familial mutant α-synuclein. These yeast gene deletions corresponded with the loss-of-function mutations seen for human DJ-1, ATP13A2, and VPS35, respectively. It was hypothesized that select gene deletions would increase certain familial mutants’ toxicity and aggregation, while leaving the rest unaffected. A potential trend showing a specific toxic interaction is presented in Figure 6. However, due to very complex nature of all of these genetic interactions, no particular gene deletion and familial mutant combo was hypothesized to become toxic. Three specific familial mutant and PD-linked gene toxicity interactions were identified in Chapter 2. H50Q and Δhsp31 was highly toxic while E46K in Δatp13 and A53T in Δvps35 were surprisingly non-toxic. 

Chapter 3

The purpose of chapter three was identical to that of Chapter 2, except for the Δvps13, Δsac1, and Δswa2 . These yeast strains correspond with the loss-of-function PD mutations seen for human VPS13C, the sac1 domain of SYNJ1, and the swa2 domain of DNAJC6, respectively. The hypothesis was identical as well. No specific familial mutant toxic interaction was found for any of the strains. However, Δvps13 broadly increased aggregation even in A30P and G51D, and Δsac1 broadly increased toxicity in WT and for all the familial mutants except A30P and G51D.

 

Figure 5. Project Design

Both WT and all six familial mutant forms were transformed into varies strains of budding yeast. Chapter one looks at α-synuclein in the context of yeast without any gene deletions (BY47471) as well as Δvps28, a strain known to increase the toxicity of WT α-synuclein. Chapter one will also look at the effects of WT α-synuclein on all eight strains used in this study. Chapter two looks at effects of WT and familial mutant α-synuclein in three yeast strains with equivalent deletions of PD-causing genes. Chapter three looks at effects of WT and familial α-synuclein in three yeast strains with equivalent deletions of PD-risk genes and domains. In all three chapters, three questions are asked and answered with three different assays.

 

Figure 6. Predicted Phenotypes in yeast with and without PD-linked gene deletions

(A) In budding yeast models, WT α-synuclein is expected to bind to the plasma membrane with minimal aggregation. The A30P and G51D mutants are expected to be cytoplasmic diffuse, while the A53T, E46K, H50Q, A53E mutants are predicted to bind to the membrane but show variable amounts of aggregation. The trend for each PD-gene deletion will vary on the gene, but there is expected to be a broad increase in aggregation in some genes but not in others. If a strong interaction is present, there should be a large increase in aggregation, shown by the E46K mutant.

(B) In budding yeast models, WT α-synuclein will be mildly toxic compared to a GFP control. The A30P and G51D mutants should appear relatively non-toxic and grow similar to the GFP control. The E46K,A53T, H50Q, and A53E mutants will be about as toxic as WT.  The trend for yeast with PD-gene deletions will vary based on the gene, but should be relatively similar to regular, BY4741 yeast. If a strong interaction is present there will be a relative reduction in growth, shown by the E46K mutant.

(C) In budding yeast models, WT α-synuclein, the E46K, A53T, H50Q, and A53E mutants should be expressed at relatively similar amounts. The A30P and G51D mutants will be expressed at relatively higher levels. The trend for yeast with a PD-gene deleted should be about the same as regular yeast, but if a strong interaction is present, there may be a large increase in protein expression, shown by the E46K mutant.

 

MATERIALS AND METHODS

The methods used in this study are based on Outeiro and Lindquist (2003), Sharma et al. (2006), and Fiske et al. (2011b). 

 

Alpha-Synuclein Constructs

A pYES2.1/V5-His-TOPO yeast expression vector is used to express α-synuclein in a budding yeast model. α-Synuclein expression is placed under the control of a GAL1 promoter region of this vector, meaning that a-synuclein protein was only expressed upon induction in galactose containing media, while being repressed upon induction in glucose containing media. α-Synuclein is bond to an eGFP tag, which co-expresses with α-synuclein and allows for α-synuclein to be visualized using GFP fluorescence microscopy.

WT-eGFP α-synuclein in pYES2.1 vector was initially provided by Dr. Virginia Lee (Pennsylvania School of Medicine). The six familial point mutations (A30P, E46K, A53T, G51D, H50Q and A53E) on WT-eGFP α-synuclein were previously generated using a GENEART site-directed mutagenesis from Invitrogen Life Technologies by multiple previous lab members. The A30P, E46K, A53T mutations were produced by Natalie Kukulka (Thesis, 2013) and the G51D, H50Q and A53E mutations were produced by Maiwase Tembo (Thesis, 2015). Mutagenesis products were transformed into One Shot™ MAX Efficiency™ DH5α™-T1R competent E. coli cells using a TOPA TA expression kit according to the included instructions. Following transformation, cells were grown on selective media (LB agar + ampicillin (50 μg/mL) to ensure that the vector was successful transformed. Mutant vector was purified from cells grown overnight in selective media (LB broth + ampicillin (50 μg/mL) using a QIAprep® Spin Miniprep Kit. Purified vector was sent to the University of Chicago DNA Sequencing Facility and confirmed to contain α-synuclein with the intended mutation and no other unintended mutations in the α-synuclein sequence. 

 

Yeast Strains 

Budding Yeast (S. cerevisiae) is used as model system for all experiments. All budding yeast strains use the BY4741, haploid background. Yeast knockout strains for yeast orthologues of PD-causing and PD-risk genes were purchased from the GE Dharmacon Yeast Knockout Collection. The yeast orthologues of disease-causing and risk genes are listed in Table 1. BY47471 with no gene deletions and vps28Δ served as controls.

 

Yeast Transformation

Both WT α-synuclein and α-synuclein containing the six familial point mutations in pYES2.1 were each transformed into all eight strains of budding yeast using a lithium acetate method from Burke et al. (2000). After transformation, budding yeast were grown in synthetic complete media lacking uracil (SC-Ura) to ensure that only yeast containing α-synuclein vector were present. All strains were also transformed with GFP-pYES 2.1 vector to serve as a non-toxic expression control.

Serial Dilution Spotting

Budding yeast cells transformed with α-synuclein were grown overnight in 5 ml SC-Ura glucose media, repressing α-synuclein expression, at 30° C and 200 rpm. Cells were collected by centrifugation at 1500 x g for 5 minutes at 4 ° C. Cells were then washed twice in 5 mL of deionized water and resuspended in to a final volume of 10 mL. Samples of cells were twice counted on a hemocytometer and a volume equivalent to 2.0 x 106 cells was determined. This volume of cell was transferred to a separate tube, pelleted, had the supernatant removed, and was suspended in 1 mL to a final concentration of 2.0 x 106 per mL. 100 µL of these cells was transferred to 96-well microtiter plate and serially diluted (1:5) five times. 2 µL of cells was then spotted onto plates containing media that induced (SC-Ura galactose) or repressed (SC-Ura glucose) α-synuclein expression. A total of three plates for each media type were used in each trial. Plates were grown at 30° until cells were visible in all six rows and imaged using an HP Canoscan Scanner and Adobe Photoshop. A spotting trial was considered acceptable if no unexpected growth was present, spotting lanes were even, and α-synuclein repressing plates had equal growth in all lanes, indicating equivalent concentrations of cells used in each row. For most strains, six trials of acceptable spotting were completed.

 

GFP Microscopy

Budding yeast cells containing α-synuclein vector were grown overnight in 5 ml SC-Ura glucose media, repressing α-synuclein expression, at 30° C and 200 rpm. Cells were collected by centrifugation at 1500 x g for 5 minutes at 4 ° C. Cells were then washed twice in 5 mL of deionized water and resuspended in to a final volume of 5 mL. A volume to of 2.0 x 107 cells transferred into a flask containing 20 mL of media that induced α-synuclein . Cells were incubated for 24 hours at 30° C and 200 rpm. Microscopy was done 6, 12, 18, 24 after initial induction in α-synuclein expressing media. At each time point 1.5 mL of cells was pelleted and 10 uL of the pelleted cells were pipetted onto a glass slide. Cells were imaged at 1000X using a TE2000-U fluorescent microscope and processed and quantified using Metamorph 4.0. For most strains, five trials of microscopy were completed.

 

Cell Scoring and Statistics

After microscopy was complete, 1000 cells for each timepoint, mutant, and strain were scored based on phenotype. The observed phenotypes are scored as appears below. Cells were scored into phenotype if each individual cell had a noticeable amount of halo-like membrane binding, cytoplasmic diffusion, or aggregation. Cells could have been classed into multiple phenotype groups if multiple phenotypes were identifiably present. A Chi-squared test of goodness-of-fit test was done to determine if each α-synuclein variant differed in terms of aggregation phenotype from the BY4741 background. A p-value of less than 0.05 was used to determine significance with a Bonferroni correction done to account for multiple mutants and timepoints being tested, resulting in a p-value of 0.002 as a cutoff point for most experiments.

 

Western Blotting Analysis

Budding yeast cells containing α-synuclein vector were grown overnight in 5 ml SC-Ura glucose media, repressing α-synuclein expression, at 30° C and 200 rpm. Cells were collected by centrifugation at 1500 x g for 5 minutes at 4 ° C. Cells were then washed twice in 5 mL of deionized water and resuspended in to a final volume of 5 mL. A volume of 2.0 x 107 cells transferred into a flask containing 20 mL of media that induced α-synuclein . Cells were incubated for 12 hours at 30° C and 200 rpm. After 12 hours, cells were counted on a hemocytometer and a volume equivalent to 2.5 x 107 cells was determined. This volume was pelleted, had the supernatant removed and was washed with 1.5 mL of a solution of 50 mM tris, 10mM sodium azide. These cells were then lysed in electrophoresis sample buffer and 10 µL was ran on a 16% Tris-glycine protein gel (Invitrogen) at 100 V using SeeBlue Pre-stained protein ladder (Invitrogen) as a standard to estimate protein size. Following electrophoresis, the gel was transferred onto a polyvinyl fluoride membrane using a semi-dry transfer technique at 15 V, for 25 minutes. The membrane was then processed with a WesternBreezeTM Chromogenic Kit (Invitrogen). Murine monoclonal antibodies were used to probe for α-synuclein (Santa Cruz Biotech) and for PGK (Invitrogen) as a loading control. Western blots were considered acceptable if each blot had one predominant band around the expected size of α-synuclein of about 65 kDa and if bands in PGK gels were appear approximately equivalent.  For most strains, two successful western blots were completed.



Strain Used

Plasmid Used

α-synuclein Construct

BY4741

pYES2.1

GFP

BY4741

pYES2.1

WT-eGFP

BY4741

pYES2.1

A30P-eGFP

BY4741

pYES2.1

E46K-eGFP

BY4741

pYES2.1

A53T-eGFP

BY4741

pYES2.1

G51D-eGFP

BY4741

pYES2.1

H50Q-eGFP

BY4741

pYES2.1

A53E-eGFP

Δvps28

pYES2.1

GFP

Δvps28

pYES2.1

WT-eGFP

Δvps28

pYES2.1

A30P-eGFP

Δvps28

pYES2.1

E46K-eGFP

Δvps28

pYES2.1

A53T-eGFP

Δvps28

pYES2.1

G51D-eGFP

Δvps28

pYES2.1

H50Q-eGFP

Δvps28

pYES2.1

A53E-eGFP

Δhsp31

pYES2.1

GFP

Δhsp31

pYES2.1

WT-eGFP

Δhsp31

pYES2.1

A30P-eGFP

Δhsp31

pYES2.1

E46K-eGFP

Δhsp31

pYES2.1

A53T-eGFP

Δhsp31

pYES2.1

G51D-eGFP

Δhsp31

pYES2.1

H50Q-eGFP

Δhsp31

pYES2.1

A53E-eGFP

Δatp13

pYES2.1

GFP

Δatp13

pYES2.1

WT-eGFP

Δatp13

pYES2.1

A30P-eGFP

Δatp13

pYES2.1

E46K-eGFP

Δatp13

pYES2.1

A53T-eGFP

Δatp13

pYES2.1

G51D-eGFP

Δatp13

pYES2.1

H50Q-eGFP

Δatp13

pYES2.1

A53E-eGFP

Δvps35

pYES2.1

GFP

Δvps35

pYES2.1

WT-eGFP

Δvps35

pYES2.1

A30P-eGFP

Δvps35

pYES2.1

E46K-eGFP

Δvps35

pYES2.1

A53T-eGFP

Δvps35

pYES2.1

G51D-eGFP

Δvps35

pYES2.1

H50Q-eGFP

Δvps35

pYES2.1

A53E-eGFP

Δvps13

pYES2.1

GFP

Δvps13

pYES2.1

WT-eGFP

Δvps13

pYES2.1

A30P-eGFP

Δvps13

pYES2.1

E46K-eGFP

Δvps13

pYES2.1

A53T-eGFP

Δvps13

pYES2.1

G51D-eGFP

Δvps13

pYES2.1

H50Q-eGFP

Δvps13

pYES2.1

A53E-eGFP

Δsac1

pYES2.1

GFP

Δsac1

pYES2.1

WT-eGFP

Δsac1

pYES2.1

A30P-eGFP

Δsac1

pYES2.1

E46K-eGFP

Δsac1

pYES2.1

A53T-eGFP

Δsac1

pYES2.1

G51D-eGFP

Δsac1

pYES2.1

H50Q-eGFP

Δsac1

pYES2.1

A53E-eGFP

Δswa2

pYES2.1

GFP

Δswa2

pYES2.1

WT-eGFP

Δswa2

pYES2.1

A30P-eGFP

Δswa2

pYES2.1

E46K-eGFP

Δswa2

pYES2.1

A53T-eGFP

Δswa2

pYES2.1

G51D-eGFP

Δswa2

pYES2.1

H50Q-eGFP

Δswa2

pYES2.1

A53E-eGFP




Table 1.  α-synuclein Constructs and Yeast Strains Used

The table above displays the yeast strains and α-synuclein constructs used in all-experiments. GFP-pYES2.1 was used as an expression control for no α-synuclein toxicity.

 

CHAPTER 1

 

CHARACTERIZATION OF A-SYNUCLEIN IN BY4741, 

 

 Δvps28, AND ACCROSS PD-LINKED GENE DELETION YEAST 

 

RESULTS



First, the phenotypes of WT and familial mutant α-synuclein were assessed in yeast without any gene deletions. This was a continuation and replication of work previously done by Natalie Kukulka (Thesis, 2013) for WT and the A30P, E46K, and A53T mutants and by Maiwase Tembo (Thesis, 2015) for WT and the G51D, H50Q and A53E mutants. To confirm the consistency and robustness of their results, select experiments were repeated and the replicated data is presented in Figures 7 and 8. Next, WT and familial mutant α-synuclein was assessed in Δvps28 yeast, which was established as mediator of α-synuclein toxicity (Willingham et al., 2003; Senagolage, Thesis, 2012). Finally, WT α-synuclein in yeast with and without the PD-gene deletions was analyzed. This was done to ensure that no PD-linked genes deletions were toxic to WT α-synuclein, except Δvps28.

 

α-Synuclein without Gene Deletions

WT and each Familial Mutant have Distinct Localizations

α-Synuclein’s normal cellular localization is disrupted in PD. Thus, if yeast is an effective model system for PD, localization phenotypes in yeasts should correspond to the cellular localization in patients. Figure 7A shows sample localization microscopy images for WT and familial mutant α-synuclein, which were quantified in Figure 7B and  Figure 7C. When WT α-synuclein was expressed in yeast, approximately 75% of cells initially showed α-synuclein membrane binding while approximately 25% of cells showed α-synuclein aggregation. Gradually α-synuclein moved to the membrane, with 90% of cells showing membrane binding by 24 hours. This corresponds to the membrane binding and mild amounts aggregation seen in sporadic PD. Approximately 90% of cells expressing either A30P or G51D α-synuclein showed cytoplasmic diffusion at all timepoints, which corresponds with previous literature and work done by Kukulka (Thesis, 2013), Tembo (Thesis, 2015), and Ong (Thesis, 2017). The E46K mutant showed somewhat more membrane-binding that WT at early timepoints, but ultimately appeared similar to WT by 24 hours and matched the results seen in Fiske et al. (2011b). The A53T and H50Q mutants had very similar localization patterns to WT. Interestingly, the A53E mutant initially had near equal amounts of membrane binding and cytoplasmic diffusion at 6 hours, but rapidly went to the membrane and appeared comparable to WT at later timepoints. Overall, WT and each mutant had distinct phenotypes that partially mimicked those exhibited in patients and corresponded with the results of previous studies.

 

WT α-Synuclein and Four Familial Mutants are Toxic, but Two are not

Given that α-synuclein toxicity is linked to cell death in PD, α-synuclein should also be toxic to yeast if it effectively models PD. To do this, the growth of yeast was assessed on growth media that either suppressed or induced α-synuclein expression. If yeast growth on the media that repressed α-synuclein was equivalent between lanes, then it was appropriate to assess the effects of α-synuclein expression media (SC-Ura galactose). Figure 8A displays the growth of WT and the six familial mutants on expression and repression media. Growth on glucose was relatively even so it was possible to evaluate the effects of α-synuclein expression. WT expression resulted in worse growth compared to the control, GFP. This result is expected as WT α-synuclein becomes toxic in cases of sporadic PD. Surprisingly, both A30P and G51D grew better than WT and showed no toxicity. This lack of toxicity may seem unexpected as these mutants cause early and aggressive forms of PD. However, these results correspond to what has been previously observed about A30P and G51D α-synuclein in yeast (Kukulka, Thesis, 2013; Tembo, Thesis, 2015). Finally, the E46K, A53T, H50Q and A53E mutants grew similar to or slightly worse than WT, consistent with prior results. 

 

α-Synuclein Expression Levels are not Linked to Toxicity

Given that PD is linked to heightened levels of α-synuclein in PD patients, heightened α-synuclein levels may also be present in and correspond to toxicity in yeast. To assess the quantity of α-synuclein present in yeast, western blotting analysis was done. An anti-α-synuclein antibody was used to measure α-synuclein levels and an anti-PGK antibody was to ensure equal amounts of yeast cells were used in each sample. Figure 8B shows protein expression levels of WT and the six familial mutants. Relatively equal PGK protein bands were present at the expected size so it possible to interpret the results of α-synuclein expression. α-Synuclein was expressed properly in WT and all familial six mutants at the expected sizes. WT α-synuclein and the E46K, A53T, H50Q, and A53E mutants were expressed at similar levels while the mutant A30P and G51D showed heightened levels of expression. This is surprising again as A30P and G51D showed cytoplasmic diffusion and were non-toxic in yeast yet expressed heightened α-synuclein levels, which should hypothetically be toxic to yeast cells. These results were slightly inconsistent with Ong (Thesis, 2017) and Tembo (Thesis, 2015), but the same conclusions were reached: the toxicity of familial mutant α-synuclein results from a mechanism independent of protein expression levels. 



α-Synuclein in Δvps28

Δvps28 Broadly Increases Aggregation 

Next, α-synuclein was expressed in Δvps28, an established mediator of α-synuclein toxicity, and the effects on localization were assessed. Quantification was not done for this set to due to time restrictions and more distinct phenotypes being present. When α-synuclein was expressed in Δvps28, broad increases in aggregation were present in WT and for the E46K, A53T, H50Q, and A53E mutants over all timepoints with prominent aggregation present even after 24 hours, as can be seen in Figure 9A. This was in stark contrast to these variants without gene deletions (BY4741) at 24 hours when only minimal aggregation was present (Figure 7A). Interestingly, the A30P and G51D mutants were unaffected and remained diffuse throughout the cell at all timepoint, implying that Δvps28 does affect these mutants and their phenotypes.

 

Δvps28 does not Alter the Trends in Toxicity 

The toxicity of α-synuclein was then assessed in Δvps28 to determine if the changes in aggregation corresponded with altered toxicity. Again, this was done by spotting analysis between GFP, WT α-synuclein, and the familial mutants in Δvps28. Glucose plates showed relatively even growth so it was possible to analyze the results in galactose media in Figure 10A. The trend for Δvps28 appeared very similar to BY4741 with WT being toxic to GFP and E46K, A53T, H50Q, and A53E having similar or slightly less growth to WT. Like BY4741, the A30P and G51D mutants were relatively non-toxic, corresponding to their diffuse phenotype. Overall, it appears that Δvps28 does not alter the trend in toxicity of the familial mutant, despite substantially increasing aggregation.

Δvps28 does not Alter the Trend in α-Synuclein Expression

Finally, α-synuclein levels for WT and the familial mutants was measured in Δvps28 by western blotting. As can be seen in Figure 10B, WT and the E46K, A53T, H50Q, and A53E mutants appeared to have relatively equal α-synuclein expression levels, while A30P and G51D showed mildly heightened expression. This overall trend matches the trend seen in yeast without gene deletions (Figure 8B). Thus, it appears that increased aggregation in Δvps28 is not due to heightened protein levels.

 

WT α-Synuclein in Yeast with and without Gene Deletions

Lastly, WT α-synuclein’s properties were compared between yeast with and without the various gene deletions that are used in this study. This was done to check if any gene deletions had broad effects that could be attributed to interactions with WT α-synuclein alone. Previous literature failed to identify any of these gene deletions as having a toxic interaction with WT α-synuclein except Δvps28 (Willingham et al., 2003; Liang et al., 2008). 

 

Δvps28, Δvps13, and Δsac1, and Δvps35 Increase Aggregation 

First, localization was assessed in each strain to determine whether any individual gene deletion resulted in increased aggregation. Figure 11A shows sample localization images for WT in each strain. Localization microscopy quantification was done only for BY4741, Δhsp31, atp13, and vps35, while the other strains were left unquantified due to time limitations. As seen before in Figure 7C, only about 25% percent of cells in BY4741 had aggregation at 6 hours which gradually decreased to about 15% by 24 hours. Δvps28, Δvps13, and Δsac1 appeared to have much higher levels of aggregation that persisted over 24 hours. The Δhsp31, Δatp13, and Δswa2 strains did not appear have any significant increases in aggregation levels at any timepoints, but the Δvps35 strain had significantly increased levels of aggregation level compared to BY47471 at all timepoints (p<0.002). 

 

Δvps28, Δatp13, Δvps35, and Δsac1 Increase Toxicity

Next, WT α-synuclein toxicity was measured between each strain to determine whether each individual gene deletion resulted in increased toxicity and if this correlated with aggregation levels. Figure 12A shows spotting results between strains. As expected, Δvps28 was substantially more toxic than BY4741. Unexpectedly, Δatp13, Δvps35, and Δsac1 showed toxicity at levels similar to Δvps28. Three of these deletions - Δvps28, Δvps35, and Δsac1 - corresponded with increased level of aggregation, but Δatp13 was toxic but did not increased aggregation, while Δvps13 was non-toxic yet showed heightened aggregation. Thus, increased aggregation may correspond with α-synuclein toxicity, but other variants, like Δatp13, must be toxic through other means. However, these results were often inconsistent between trials, and thus require more repetition to confirm the exact effects of these gene deletions.

 

Toxicity and Aggregation is not due to Increased Protein Levels

Finally, protein expression was analyzed for WT α-synuclein in each strain to determine if each gene-deletion’s toxicity or localization could be attributed to heightened protein expression. Figure 12B shows WT α-synuclein’s expression between strain. WT α-synuclein’s expression varied only mildly between each strain and this variation could potentially be attributed to inconsistent PGK protein levels. Furthermore, the strains that had either increased aggregation and toxicity, Δvps28, Δvps35, Δatp13 and Δsac1, did not have heightened protein expression. Thus, protein expression does not seem to be related to aggregation or localization for WT α-synuclein between these strains.

 

Figure 7. Localization α-Synuclein in BY4741

(A) Microscopy images show the localization phenotypes for WT and familial mutant α-synuclein. Images were captured at 6, 12, 18, and 24 hours post induction in α-synuclein expressing media (SC-Ura galactose) (n=5).

(B) Microscopy images for 1000 cells of WT and each familial mutant were quantified based on which α-synuclein localization phenotypes are present. The error bars present represent standard error (n=4).

(C) Microscopy images for 1000 cells of WT and each familial mutant were quantified based on which α-synuclein localization phenotypes are present with combined phenotypes integrated into the cells with halo, diffuse, or aggregation phenotypes. Total percentage may exceed 100% due to a portion of cells falling in multiple phenotypic categories. The error bars present represent standard error (n=4).

 

Figure 8. Toxicity and Protein Expression of α-Synuclein in BY4741

(A) Five-fold serial dilution spotting of yeast expressing WT and familial mutant α-synuclein spotted onto α-synuclein repressing (SC-Ura Glucose) and expressing media (SC-Ura Galactose) (n=6).

(B) Western Blot analysis of yeast expressing WT and familial mutant α-synuclein 12 hours post induction in α-synuclein expressing media (SC-Ura galactose). Protein expression was observed using an anti-α-synuclein antibody and an anti-PGK antibody as a loading control (n=4).

 

Figure 9. Localization of α-Synuclein in Δvps28

(A) Microscopy images show the localization phenotypes for WT and familial mutant α-synuclein. Images were captured at 6, 12, 18, and 24 hours post induction in α-synuclein expressing media (SC-Ura galactose) (n=5).

 

Figure 10. Toxicity and Protein Expression of α-Synuclein in Δvps28

(A) Five-fold serial dilution spotting of yeast expressing WT and familial mutant α-synuclein spotted onto α-synuclein repressing (SC-Ura Glucose) and expressing media (SC-Ura Galactose) (n=6).

(B) Western Blot analysis of yeast expressing WT and familial mutant α-synuclein 12 hours post induction in α-synuclein expressing media (SC-Ura galactose). Protein expression was observed using an anti-α-synuclein antibody and an anti-PGK antibody as a loading control (n=2).

 

Figure 11. Localization of WT α-Synuclein between Strains

(A) Microscopy images show the localization phenotypes for WT α-synuclein in BY4741, Δvps28, Δ hsp31, Δatp13, Δvps35, Δvps13, Δsac1, and Δswa2 strains. Images were captured at 6, 12, 18, and 24 hours post induction in α-synuclein expressing media (SC-Ura galactose) (n=5).

 

Figure 12. Toxicity and Protein Expression between Strains

(A) Five-fold serial dilution spotting of yeast expressing WT α-synuclein in BY4741, Δvps28, Δ hsp31, Δatp13, Δvps35, Δvps13, Δsac1, and Δswa2 strains spotted onto α-synuclein repressing (SC-Ura Glucose) and expressing media (SC-Ura Galactose) (n=6).

(B) Western Blot analysis of yeast expressing WT α-synuclein in BY4741, Δvps28, Δhsp31, Δatp13, Δvps35, Δvps13, Δsac1, and Δswa2 strains 12 hours post induction in α-synuclein expressing media (SC-Ura galactose). Protein expression was observed using an anti-α-synuclein antibody and an anti-PGK antibody as a loading control (n=2).

 

 

 

CHAPTER 2

 

CHARACTERIZATION OF A-SYNUCLEIN IN

 

 Δhsp31, Δatp13, and Δvps35

 

 

 α-Synuclein in PD-Causing Gene Deletion Strains

Now that both WT and familial mutant α-synuclein had been analyzed in yeast without gene deletions (BY4741) and between yeast with and without gene deletions, it was possible determine the interaction between each familial mutant and each PD-linked gene deletion. In this section, WT and familial mutant α-synuclein localization, toxicity, and protein expression trends were assessed for Δhsp31, Δatp13, Δvps35. These trends were then compared to the trends for yeast without gene deletions (BY4741) that were seen in Figure 7 and Figure 8.

 

α-Synuclein in Δhsp31

Δhsp31 Specifically Increases H50Q aggregation

First, WT and familial mutant α-synuclein was assessed in Δhsp31 to determine if Δhsp31 alters any of the localization patterns for familial mutant α-synuclein. Figure 13A shows sample localization microscopy images for WT and familial mutant α-synuclein in Δhsp31, which was quantified in Figure 13B and Figure 13C. Approximately 25% cells with WT α-synuclein showed aggregation at 6 hours which gradually decreased to about 15% by 24 hours, which was similar to BY4741(Figure 7C). No mutant had any major alteration to localization phenotype except H50Q. H50Q had a nearly twofold increase in cells showing α-synuclein aggregation, with approximately 55% aggregation at 6 hours and 35% aggregation by 24 hours (Figure 13C). H50Q aggregation was significantly higher in Δhsp31 for all of timepoints (p<0.002). 

 

H50Q is Highly Toxic in Δhsp31

Next, toxicity was assessed in Δhsp31. This was done to determine if a specific familial mutant was toxic in Δhsp31 and if this toxicity was related to aggregation. Figure 14A displays the spotting results for Δhsp31. WT α-synuclein was toxic compared to GFP, similar to BY4741 (Figure 8A). Both A30P and G51D were relatively non-toxic and E46K, A53T, and A53E showed similar toxicity to WT, again like BY4741. However, H50Q was specifically and uniquely toxic in Δhsp31, as can be seen by its reduced growth in Figure 14A. Thus both, aggregation and toxicity are increased in Δhsp31 for the H50Q mutant. 

 

H50Q Aggregation and Toxicity is not Due to Increased Protein Expression

Finally, α-synuclein expression was compared in Δhsp31. This was done to determine if H50Q’s aggregation and toxicity was due to increased α-synuclein levels. Figure 14B shows protein expression levels for WT and the six familial mutants. WT, E46K, A53T, and A53E expressed equivalent levels of α-synuclein while A30P and G51D showed heightened expression. This corresponds to the trend seen in BY4741 (Figure 8B). H50Q expression appeared to be increased slightly, with levels similar to A30P or G51D (Figure 14B). This slight increase in expression did not appear to be sufficient to account for the massive increase in aggregation and toxicity for H50Q. Thus, H50Q’s increased aggregation and toxicity appears to be due to mechanisms independent of α-synuclein levels.

 

α-synuclein in Δatp13

Δatp13 has no Significant Effect on Localization 

Like Δhsp31, localization in Δatp13 was analyzed first. Figure 15A shows sample localization images in Δatp13 which are quantified in Figure 15B and Figure 15C. WT α-synuclein had about 25% percent of cells with aggregation at 6 hours which gradually decreased to about 12% by 24 hours with most cells showing membrane binding. A30P and G51D were diffuse throughout the time course with all timepoints showing near 90% cytoplasmic diffusion. The E46K, A53T, H50Q, and A53E mutants showed similar trends to BY4741 with no mutant showing significant difference in aggregation at any timepoint. No obvious alterations in localization were observed in Δatp13 when compared to BY4741 (Figure 7A)

 

E46K α-Synuclein is Non-Toxic in Δatp13 

Next, the effects of Δatp13 on toxicity was measured. This is presented in Figure 16A. As expected, WT α-synuclein was toxic relative to GFP. Both A30P and G51D were less toxic than WT. A53T, H50Q, and A53E showed toxicity similar to or slightly worse than WT. Surprisingly, E46K was non-toxic to levels similar to A30P or GFP, in contrast to its usually moderate presentation in BY4741(Figure 8A). This effect may be due to its near complete membrane binding present in all timepoints in microscopy as seen in Figure 15C.

 

E46K Expression is Reduced in Δatp13

Finally, protein expression was assessed in Δatp13 . This was done to determine E46K’s non-toxic phenotype correlated with reduced expression. Figure 16B shows α-synuclein levels in Δatp13. The overall trend for WT and five mutants was similar to that of BY4741(Figure 8B). WT, A53T, H50Q, and A53E showed similar expression while A30P and G51D showed heightened expression.  However, E46K showed considerably reduced expression compared to WT and appeared greatly reduced compared to its expression in BY4741. Thus, E46K’s non-toxic phenotype is likely due to reduced protein expression and potentially due to reduced aggregation and increased membrane binding.

 

α-Synuclein in Δvps35

Δvps35 Broadly Increases Aggregation, Except for A53T which is Diffuse

Finally, α-synuclein localization was assessed in Δvps35. Figure 17A shows sample microscopy in Δvps35 that is quantified in Figure 17B and Figure 17C. α-Synuclein aggregation was significantly increased in WT and H50Q for all timepoints and at 6 and 12 hours for A53E compared to BY4741 (p<0.002). A30P, G51D and E46K appeared unaffected and showed localization similar to BY4741(Figure 7A). A30P and G51D appeared cytoplasmically diffuse and E46K was tightly bound to the membrane at all timepoints. Surprisingly, A53T in Δvps35 was overwhelmingly cytoplasmically diffuse at all timepoints, in stark contrast to its usual membrane binding and mild aggregation seen in BY4741. This phenotype has never previously been observed for A53T and thus required further investigation. 

 

A53T is Non-toxic in Δvps35 

To continue the investigation of Δvps35, the α-synuclein toxicity was assessed and is presented in Figure 18A. As seen before with Δvps35 in Figure 12A, WT α-synuclein was particularly toxic compared to GFP in Δvps35. E46K, H50Q, and A53E displayed similar levels of toxicity to WT and the A30P and G51D mutants were relatively non-toxic. Interestingly, A53T lacked toxicity and grew similarly to A30P or the GFP control. Thus, A53T’s diffuse phenotype localization appears linked to a lack of toxicity in Δvps35.

 

A53T’s Expression is Reduced in Δvps35 

Lastly, α-synuclein expression analysis was done in Δvps35 and is presented in Figure 18B. WT and the A30P, E46K, G51D, H50Q, and A53E mutants showed expression patterns similar to BY4741(Figure 8B). However, A53T had slightly reduced expression levels compared to the trend seen in BY4741. Thus, it seems that reduced protein expression may be responsible for A53T’s lack of toxicity and diffuse phenotype in Δvps35, but more western analysis is required to confirm this effect. The phenotypes seen for A53T in Δvps35 can be contrasted to what is seen for A30P and G51D. A30P, G51D, and A53T in Δvps35 are all non-toxic and cytoplasmically diffuse, but A30P and G51D show elevated expression while A53T shows reduced expression.

 

Figure 13. Localization of α-Synuclein in Δhsp31

(A) Microscopy images show the localization phenotypes for WT and familial mutant α-synuclein. Images were captured at 6, 12, 18, and 24 hours post induction in α-synuclein expressing media (SC-Ura galactose) (n=5).

(B) Microscopy images for 1000 cells of WT and each familial mutant were quantified based on which α-synuclein localization phenotypes are present. The error bars present represent standard error (n=4).

(C) Microscopy images for 1000 cells of WT and each familial mutant were quantified based on which α-synuclein localization phenotypes are present with combined phenotypes integrated into the cells with halo, diffuse, or aggregation phenotypes. Total percentage may exceed 100% due to a portion of cells falling in multiple phenotypic categories. The error bars present represent standard error (n=4).

 

Figure 14. Toxicity and Protein Expression of α-Synuclein in Δhsp31

(A) Five-fold serial dilution spotting of yeast expressing WT and familial mutant α-synuclein spotted onto α-synuclein repressing (SC-Ura Glucose) and expressing media (SC-Ura Galactose) (n=6).

(B) Western Blot analysis of yeast expressing WT and familial mutant α-synuclein 12 hours post induction in α-synuclein expressing media (SC-Ura galactose). Protein expression was observed using an anti-α-synuclein antibody and an anti-PGK antibody as a loading control (n=2).

 

Figure 15. Localization of α-Synuclein in Δatp13

(A) Microscopy images show the localization phenotypes for WT and familial mutant α-synuclein. Images were captured at 6, 12, 18, and 24 hours post induction in α-synuclein expressing media (SC-Ura galactose) (n=5).

(B) Microscopy images for 1000 cells of WT and each familial mutant were quantified based on which α-synuclein localization phenotypes are present. The error bars present represent standard error (n=4).

(C) Microscopy images for 1000 cells of WT and each familial mutant were quantified based on which α-synuclein localization phenotypes are present with combined phenotypes integrated into the cells with halo, diffuse, or aggregation phenotypes. Total percentage may exceed 100% due to a portion of cells falling in multiple phenotypic categories. The error bars present represent standard error (n=4).

 

Figure 16. Toxicity and Protein Expression of α-Synuclein in Δatp13

(A) Five-fold serial dilution spotting of yeast expressing WT and familial mutant α-synuclein spotted onto α-synuclein repressing (SC-Ura Glucose) and expressing media (SC-Ura Galactose) (n=6).

(B) Western Blot analysis of yeast expressing WT and familial mutant α-synuclein 12 hours post induction in α-synuclein expressing media (SC-Ura galactose). Protein expression was observed using an anti-α-synuclein antibody and an anti-PGK antibody as a loading control (n=2).

 

Figure 17. Localization of α-Synuclein in Δvps35

(A) Microscopy images show the localization phenotypes for WT and familial mutant α-synuclein. Images were captured at 6, 12, 18, and 24 hours post induction in α-synuclein expressing media (SC-Ura galactose) (n=5).

(B) Microscopy images for 1000 cells of WT and each familial mutant were quantified based on which α-synuclein localization phenotypes are present. The error bars present represent standard error (n=4).

(C) Microscopy images for 1000 cells of WT and each familial mutant were quantified based on which α-synuclein localization phenotypes are present with combined phenotypes integrated into the cells with halo, diffuse, or aggregation phenotypes. Total percentage may exceed 100% due to a portion of cells falling in multiple phenotypic categories. The error bars present represent standard error (n=4).

 

Figure 18. Toxicity and Protein Expression of α-Synuclein in Δvps35

(A) Five-fold serial dilution spotting of yeast expressing WT and familial mutant α-synuclein spotted onto α-synuclein repressing (SC-Ura Glucose) and expressing media (SC-Ura Galactose) (n=6).

(B) Western Blot analysis of yeast expressing WT and familial mutant α-synuclein 12 hours post induction in α-synuclein expressing media (SC-Ura galactose). Protein expression was observed using an anti-α-synuclein antibody and an anti-PGK antibody as a loading control (n=2).

 

CHAPTER 3

 

CHARACTERIZATION OF A-SYNUCLEIN IN

 

 Δvps13, Δsac1, and Δswa2

 

 α-Synuclein in PD-Risk Gene Deletion Strains

The experimental purpose for the PD-risk gene deletion strains was identical to that for the PD-causing gene deletion strains: to identify alterations in phenotypic trends between each strain and BY4741. Δvps13, Δsac1, and Δswa2 are assessed in this section which correspond to human VPS13C, the sac1 domain of human SYNJ1, and the swa2 domain of human DNAJC6. Localization, toxicity, and protein expression were assessed for each strain but localization quantification analysis was not conducted due to time limitations.

 

α-Synuclein in Δvps13

Δvps13 Broadly Increases Aggregation, even in A30P and G51D

Localization microscopy was done first in Δvps13. Sample microscopy images are presented in Figure 19A. WT α-synuclein in Δvps13 showed both membrane binding and aggregation that persisted over 24 hours. A53T, H50Q, and A53E showed increased aggregation like WT while E46K maintained its overwhelmingly membrane bond phenotype. The A30P and G51D mutants also maintained their cytoplasmically diffuse phenotypes but additionally had substantial amounts of aggregation that remained present even at 24 hours. This combined aggregation-diffusion phenotype is unique to A30P and G51D in Δvps13 and hasn’t been seen previously in any of the strains with any forms of α-synuclein.

 

Δvps13 has Little Effect on Familial Mutant Toxicity

Next, toxicity was measured in Δvps13 to determine if the broad increase in aggregation resulted in a specific increase in toxicity among the familial mutants, particularly for A30P and G51D. As can be seen in Figure 20A, WT α-synuclein is toxic to WT as expected. The E46K, A53T, H50Q, and A53E mutants appeared just as toxic to WT. The A30P and G51D mutants were relatively non-toxic, with G51D potentially showing mild toxicity compared A30P. This largely matches that trend seen for BY4741 (Figure 8B).  No particularly mutant appeared to be substantially toxic in in Δvps13 despite major changes in localization.

 

Δvps13 has Little Effect on Expression Levels

Finally, α-synuclein protein levels were measured in Δvps13 and results are presented in Figure 20B. WT, E46K, A53T, H50Q, and A53E showed similar expression levels with A30P and G51D having slightly heightened protein levels. These trends matched BY4741 (Figure 8B). Thus, it appears that Δvps13 does not affect familial mutant toxicity and protein levels, despite substantially increasing aggregation.

 

α-Synuclein in Δsac1

Δsac1 Mildly Increases Aggregation

WT and the familial mutants were next evaluated in Δsac1 and sample localization images are presented in Figure 21A. WT α-synuclein had both membrane binding and aggregation with substantial aggregation still present at 24 hours. E46K, A53T, H50Q, and A53E appeared to have increased aggregation at early timepoints, but looked more like BY4741 by 24 hours with less aggregation and more membrane binding. The A30P and G51D mutants appeared cytoplasmically diffuse throughout 24 hours with small amounts of aggregation at early timepoints for G51D. Overall, it appears that Δsac1 increases WT aggregation but not have any familial mutant specific effect.

Δsac1 is Broadly Toxic to WT and Four Familial Mutant

Next, toxicity was assessed in Δsac1 and spotting results are presented in Figure 22A. WT α-synuclein was particularly toxic in Δsac1 compared to the GFP control. The E46K, A53T, H50Q, and A53E mutants showed toxicity a similar levels of toxicity . The A30P and G51D mutants appeared relatively non-toxic. The overall phenotype trend matched that seen in BY4741 (Figure 8A) with all mutants showing increased toxicity except A30P and G51D.

 

Δsac1 Toxicity Appears to not be Linked to α-synuclein Expression

Finally, α-synuclein protein levels were assessed in the Δsac1 to determine if Δsac1’s broad toxicity was related to altered protein expression. Figure 22B shows the relative α-synuclein in Δsac1. WT, E46K, A53T, H50Q, and A53E α-synuclein showed relatively even levels of expression while the A30P and G51D had slightly elevated expression compared to WT. Overall this corresponded to BY4741’s expression pattern (Figure 8B) and it appears that WT, E46K, A53T, H50Q, and A53Es’ toxicity in Δsac1 is not due heightened protein levels.



α-synuclein in Δswa2

Δswa2 has Little Effect on Localization

The last PD-linked gene deletion that α-synuclein was in assessed was Δswa2. Sample Localization images are presented in Figure 23A. WT had small amounts aggregation at early timepoints but α-synuclein mostly membrane bound by 18 and 24 hours. A30P and G51D showed diffusion at all timepoints, although their diffuse phenotype had a morphology distinct from what is normally seen in BY4741(Figure 7A). E46K, A53T, H50Q, and A53E also had with mild amounts of aggregation and membrane binding similar to BY4741. Overall, Δswa2 has very little effect on α-synuclein localization, particularly aggregation. 

 

Δswa2 has Little Effect on α-Synuclein Toxicity.

Next, the toxicity of α-synuclein in Δswa2 was assessed. As can be seen in Figure 24A, WT α-synuclein is mildly toxic compared to GFP alone. Both, A30P and G51D had were non-toxic while E46K, A53T, H50Q, and A53E showed similar toxicity to WT. These phenotypes match those seen in BY4741 almost identically (Figure 24A). Thus, Δswa2 does not alter toxicity of the familial mutants or WT.

 

Δswa2 has Little Effect on α-Synuclein Protein Expression.

The last experiment done in Δswa2 was protein expression analysis. As can be seen in Figure 24B, WT, E46K, A53T, H50Q, and A53E showed equivalent protein expression while A30P and G51D had elevated expression. These results correspond almost exactly with those present in BY4741 (Figure 8B). Overall it appears that Δswa2 did not alter any of α-synuclein’s properties in yeast that were measured.

 

Figure 19. Localization of α-Synuclein in Δvps13

(A) Microscopy images show the localization phenotypes for WT and familial mutant α-synuclein. Images were captured at 6, 12, 18, and 24 hours post induction in α-synuclein expressing media (SC-Ura galactose) (n=5).

 

Figure 20. Toxicity and Protein Expression of α-Synuclein in Δvps13

(A) Five-fold serial dilution spotting of yeast expressing WT and familial mutant α-synuclein spotted onto α-synuclein repressing (SC-Ura Glucose) and expressing media (SC-Ura Galactose) (n=6).

(B) Western Blot analysis of yeast expressing WT and familial mutant α-synuclein 12 hours post induction in α-synuclein expressing media (SC-Ura galactose). Protein expression was observed using an anti-α-synuclein antibody and an anti-PGK antibody as a loading control (n=2)

 

Figure 21. Localization of α-Synuclein in Δsac1

(A) Microscopy images show the localization phenotypes for WT and familial mutant α-synuclein. Images were captured at 6, 12, 18, and 24 hours post induction in α-synuclein expressing media (SC-Ura galactose) (n=5).

 

Figure 22. Toxicity and Protein Expression of α-Synuclein in Δsac1

(A) Five-fold serial dilution spotting of yeast expressing WT and familial mutant α-synuclein spotted onto α-synuclein repressing (SC-Ura Glucose) and expressing media (SC-Ura Galactose) (n=6).

(B) Western Blot analysis of yeast expressing WT and familial mutant α-synuclein 12 hours post induction in α-synuclein expressing media (SC-Ura galactose). Protein expression was observed using an anti-α-synuclein antibody and an anti-PGK antibody as a loading control (n=2)

 

Figure 23. Localization of α-Synuclein in Δswa2

(A) Microscopy images show the localization phenotypes for WT and familial mutant α-synuclein. Images were captured at 6, 12, 18, and 24 hours post induction in α-synuclein expressing media (SC-Ura galactose) (n=5).

 

 Figure 24. Toxicity and Protein Expression of α-Synuclein in Δswa2

(A) Five-fold serial dilution spotting of yeast expressing WT and familial mutant α-synuclein spotted onto α-synuclein repressing (SC-Ura Glucose) and expressing media (SC-Ura Galactose) (n=6).

(B) Western Blot analysis of yeast expressing WT and familial mutant α-synuclein 12 hours post induction in α-synuclein expressing media (SC-Ura galactose). Protein expression was observed using an anti-α-synuclein antibody and an anti-PGK antibody as a loading control (n=2)

 

DISCUSSION

 

Parkinson’s disease is a complex neurodegenerative disease influenced by multiple environmental and genetic factors. While the etiology for most cases of PD remains unknown, three fundamental characteristics are present in nearly all cases of PD: motor impairment, selective dopaminergic SNpc degeneration, and α-synuclein aggregation and toxicity. Even though familial PD only accounts for about 10-15% of total cases, the molecular mechanisms of toxicity that underlie many cases of familial PD – such as oxidative stress, impaired protein and vesicular transport, or membrane instability – are often identical the same in sporadic PD. Familial PD genes include DJ-1, ATP13A2, VPS35, and SNCA, the last which codes α-synuclein and has six disease causing point mutations. The functions of many of these genes are well understood. However, their interactions with α-synuclein, which is arguably more important for PD, is less so.

 Thus, the overall purpose of this thesis was to identify interactions between yeast equivalents of six-PD linked genes and α-synuclein. Another goal was to identify if these genes had specific interactions with familial mutant α-synuclein with the hope of better understanding each familial mutant’s specific mode of toxicity. It was hypothesized that select gene deletions to would increase α-synuclein aggregation and toxicity in a familial mutant specific manner, while others would have no effect. 

 

Each PD-linked Gene Deletion has Specific Effect on α-synuclein

Overall, this hypothesis was partially supported. The clearest support came from the H50Q mutant in Δhsp31, which was specifically and highly toxic and had heightened aggregation. Other support for this hypothesis came from a lack specific familial mutant toxicity in Δvps13, Δsac1, and Δswa2. 

However, there was substantial evidence that deletions had non-toxic or non-specific effects. Three unexpected gene deletions, Δatp13, Δvps35, and Δsac1 increased WT α-synuclein to toxicity to levels equivalent to Δvps28, a previously established moderator of toxicity. However, this set of experiments provided highly inconsistent results. Consequently, it cannot be confidently concluded that these strains are all toxic to WT. Furthermore, the Δvps35, Δvps13, and Δsac1 mutants showed broad increases in aggregation for WT and multiple mutants, but only two showed increased toxicity. Additionally, specific non-toxic interactions were present for E46K in Δatp13 and A53T in Δvps35. This is highly unexpected as this is equivalent of two-disease causing mutations combining to produce a non-toxic effect. Overall, evidence from this thesis indicates that each PD-linked gene deletion has a unique effect on α-synuclein’s properties. This effect can be either toxic or protective and broad or mutant specific. 

 

H50Q Toxicity

As stated before, the strongest support for the familial mutant-specific hypothesis came from results for H50Q in Δhsp31, which was highly toxic and showed increased aggregation. Multiple potential modes of toxicity have been proposed and experimentally supported for H50Q, including altered copper binding, increased aggregation, mitochondrial impairment, and increased oxidative stress but the mechanism is unknown (Khalaf et al., 2014; Ghosh et al., 2013; Villar-Piqué et al., 2017). H50Q’s specific increase in toxicity and aggregation without heightened protein expression in Δhsp31 provides support that this mutant becomes toxic when oxidative stress is increased and mitochondria are damaged, which result from the loss of hsp31’s normal function (Junn et al., 2009; Aslam and Hazbun, 2016). The corresponds with previous work which showed that H50Q expressing neuronal cultures were particularly sensitive to increased oxidative stress compared to WT cells (Xiang et al., 2015). To further determine the exact nature of this effect, mitochondria structure and oxidative stress levels in yeast could be observed and compared between WT and H50Q in both Δhsp31 and BY4741 using immunofluorescence microscopy with antibodies targeting mitochondria and oxidative stress products (Elliott and Volkert, 2004). Another potential method to the determine the nature of H50Q toxicity would be to introduce human DJ-1 into yeast using a similar vector system done in this study (Osborn and Miller, 2007). If DJ-1 introduction rescued the toxicity seen for H50Q in Δhsp31, it would help show that DJ-1’s function is conserved in yeast hsp31 and that this toxicity is mediated by mitochondrial impairment and oxidative stress.

 

Two Non-toxic Mutants in Two Strains?

The protective interactions between E46K and Δatp13 and between A53T and Δvps35 were highly unexpected and are somewhat contrary to what has been found in previous literature. E46K’s proposed mechanism of toxicity is thought to function through altered membrane association or fibrilization, but not through abnormal metal transport which might be altered by deletion of atp13 (Íñigo-Marco et al., 2017; Schmidt et al., 2009). Published research looking at E46K in the context of metal interactions or genetic interactions with ATP13A2 is extremely limited. E46K binds to copper and iron differently than WT and that increased copper binding may alter interactions specific to E46K’s structure between the N- and C- terminuses (Bharathi and Rao, 2008; Rospigliosi et al., 2009). However, yeast atp13 gene does not appear to interact with copper or iron so this non-toxic interaction is still unexplainable (van Veen et al., 2014; Schmidt et al., 2009).  Like E46K, A53T’s protective interaction with Δvps35 was highly unexpected. If anything, A53T should be more toxic in Δvps35. VPS35 is responsible for post-ER retrograde transport and protein degradation and A53T toxicity should be compounded if it cannot be degraded (Miura, et al., 2014; Conway et al., 1998; Sharon et al., 2015). Hypothetically, Δvps35 should result in less degradation, as it does in all other mutants who showed heightened aggregation in this study. Again literate [sic] looking at the interaction between A53T and VPS35 is limited. VPS35 was shown to reduced degradation of A53T when VPS35 is mutated in mammalian cell culture (Zavodszky et al., 2014). Additionally, A53T expressions impairs proper synapse organization more than WT α-synuclein in C. elegans with vps35 mutations. Thus, A53T becoming non-toxic in Δvps35 is still unexplained. 

 

Cytoplasmic Diffusion in Yeast is Non-toxic

Interestingly, A53T in Δvps35 also showed cytoplasmic diffusion and was non-toxic. This matches phenotypes for both A30P and G51D, which nearly always have cytoplasmic diffusion and corresponding with non-toxicity across multiple strains of yeast with gene deletions (Kulkulka, Thesis 2013, ; Fares et al., 2014; Tembo, Thesis, 2015). This non-toxic effect was also mirrored in yeast with other point mutations that resulted in cytoplasmically diffuse phenotype (Ong, Thesis, 2017).  This study further showed that A30P and G51D maintain their non-toxic effects even if they show a combined diffuse and aggregated phenotype, as seen in Δvps13. Thus, it is reasonable to conclude that some characteristic about the cytoplasmically diffuse phenotype protects yeast from α-synuclein toxicity. Since A30P and G51D have shown the most consistent cytoplasmically diffuse phenotype, there must be some characteristic about these two mutants that is responsible for their effects. Both A30P and G51D are unique in that they disrupt an N-terminal alpha-helix that normally anchors α-synuclein to the cell membrane, resulting in a cytoplasmically diffuse phenotype (Fares et al., 2014; Jensen et al., 1998). They also both show significant reductions in aggregation while not affecting oligomerization compared when compared to the E46K, A53T, and H50Q mutants (Lesage et al., 2013a, Sierecki et al., 2016). However, G51D does not behave identical to A30P as it aggregates slower, results in nuclear localization and increased mitochondrial impairment, and causes much earlier onset of PD. The loss of membrane binding may underlie this non-toxic effect as proposed by Brandis et al. (2006) and Sharma et al. (2006), but this conflicts with results seen in E46K in Δatp13 which is non-toxic yet is extremely tightly bound to the membrane. Clearly, the mechanisms of toxicity for A30P and G51D toxicity in humans are distinct from WT and the other four mutants. It is still unknown why these particularly mutants and the cytoplasmically diffuse phenotype is non-toxic to yeast.

  

Mutant Toxicity is not Due to Increased Protein Accumulation

One mechanism of toxicity that does not appear to be involved in any familial mutant or gene deletion is α-synuclein proteins. Throughout most experiments and between multiple PD-linked gene deletion strains, protein levels between WT α-synuclein and the six mutants did not vary substantially, and if they did, the non-toxic mutants, A30P and G51D, generally had elevated protein levels. This slightly conflicts with results seen by both Tembo (Thesis, 2015) and Ong(Thesis, 2017) who failed to show heightened G51D expression compared WT but corresponds with heightened expression seen by Sharma et al., (2006) for A30P tagged with GFP. However, both Ong and Tembo showed inconsistent levels of G51D expression between experiments and used different timepoints to measure protein levels than were done in this thesis, so it possible that the data between studies may differ.  In most experiments and strains in this study, the A30P and G51D mutants had slightly increased levels of α-synuclein that corresponded with a non-toxic phenotype. This may seem counterintuitive as increased levels of α-synuclein are broadly implicated in PD and mutations that increase α-synuclein expression cause familial PD (Chartier-Harlin et al., 2004; Singleton et al., 2003). However, there is substantial evidence that α-synuclein oligomerization and modification may be more important to toxicity than α-synuclein levels alone (Soper et al., 2008; Burré et al. 2015). A30P and G51D α-synuclein may accumulate more in yeast, but remain non-toxic due to the lack of the formation of larger oligomerizes or through other unidentified mechanisms. There is limited evidence that reduced toxicity corresponds with reduced protein levels as seen for both E46K in Δatp13 and A53T in Δvps35, but it is difficult to generalize these results.  These results are isolated, have unexpected non-toxic effects, show opposite microscope phenotypes, and have few complete trials of western blotting analysis (n=2). Together, these results indicate that altered protein expression is not a strong indicator or mediator of familial α-synuclein toxicity or toxicity due to PD-linked gene deletion.

 

Three New Toxic Gene Deletions

This study also identified three potential new genes that may mediate WT α-synuclein toxicity - Δatp13, Δvps35, Δsac1. Previous genetic screens failed to find that these gene deletions increased α-synuclein nor were any found to mediate familial mutant toxicity (Willingham et al., 2003; Liang et al., 2008). However, both studies used different strains of budding yeast from the current one and both utilized a lower α-synuclein expression system. It may be that these gene deletions only increase α-synuclein toxicity when it is at a level that α-synuclein induces toxicity itself.  Additionally, two of these strains showed increased aggregation that corresponded with increased toxicity for WT α-synuclein - Δvps35, Δsac1 – while Δatp13 did not show increased aggregation yet was still toxic and Δvps13 showed increased aggregation but did not increase toxicity. This affirms that toxicity can be derived from impaired protein trafficking as seen by aggregation that can result from Δvps35, Δsac1, or Δvps28 or through other means as seen in Δatp13 (Seaman, 2012; McPherson et al., 1996; Schmidt et al., 2009). Additionally, it appears that only specific vps deletions are toxic even if they increase aggregation yeast as seen by Δvps13 and previous work exploring multiple vps deletions (Senagolage, Thesis, 2012). Overall, this supports the hypothesis that specific genes facilitate WT α-synuclein toxicity through different mechanisms while others do not contribute to this effect.

 

Limitations of this Study

While budding yeast have provided interesting and significant results for this study, there are considerations to take into account before making a broad conclusion. While yeast do show similar α-synuclein localization phenotypes to neurons, they are structurally different to neurons and do not release neurotransmitters, which is impaired in PD. Yeast also replicate multiple times during a 24-hour period while neurons do not replicate at all and can survive for decades. Additionally, yeast do not natively express α-synuclein so any effect present in yeast is specific to α-synuclein and not necessarily reflective of what is happening in the brains of patients. The vector expression system in this study also expressed a higher level of α-synuclein to many studies done previously, so it may be easier to find significant results that may not be physiologically relevant when compared to earlier studies. Budding yeast also often shows distinct phenotypes to other types yeast, such as fission yeast, upon induction with α-synuclein (Brandis et al., 2006)

While yeasts have many of the same functional genes as humans, there is relatively little sequence homology between human and yeast genes so there may be meaningful differences in how they function. Full yeast gene deletions do mimic the effects of many of the mutations present in the PD-linked genes analyzed. However, many other mutations have other gain-of-function effect while others have only partial loss-of-function effects that aren’t accurately mimicked by full gene deletions. Lastly, yeast genes often have multiple orthologues in humans and deletion of one yeast gene may correspond to the loss of multiple human genes. This can be clearly seen for Δvps13 which not only corresponds to the deletion of human PD-related VPC13C but also to be VPS13A, VPS13B, and VPS13D. VPS13A and VPS13D have not been linked to be PD but are linked to other unrelated disorders such as Chorea-acanthocytosis and Cohen’s Syndrome (Muñoz-Braceras et al., 2015; Balikova et al., 2009). It is essential to understand these limitations before making conclusions about any of the results in this study.

 

Future Research

While the overwhelming bulk of bench research for this project has been completed, there are a few areas where replications are necessary. Western Blotting analysis for most strains had only two trials completed. Conclusions drawn from these results would be stronger if replicated several more times. Additionally, the results of Figure 12A were very inconsistent so it is difficult to make proper conclusions about WT toxicity between strains. Higher quality replications are necessary for this experiment to make any meaningful conclusions about toxicity.

To help combat some of the limitations of this study, it would be beneficial to determine the similarities in differences between the human and yeast genes assessed. This could be done by putting the human equivalent of the lost yeast gene back into the yeast and observing if the phenotypes are reversed back. This would provide better evidence that the specific function of each human version of the PD-linked gene is responsible for the familial mutant specific effect rather than just the less specific effects of the lost yeast gene.

One avenue of future research would look more at the specific nature of α-synuclein accumulation and aggregation in the various familial mutants. Since α-synuclein oligomerization is more strongly linked to toxicity than protein, it may be beneficial to look at levels of oligomers and aggregates present in yeast and determine if this correlates with increased toxicity in spotting and aggregation in microscopy. This can be done using either size exclusion chromatography or sucrose gradient fraction, both of which have shown that certain modification can mediate these effects (Vicente Miranda et al., 2017). This could be used to potentially explain the non-toxic effect seen for A30P and G51D as well as for E46K in Δatp13 and A53T in Δvps35, if they may show fewer oligomers than WT. 

 

Conclusions:

My Findings

Overall, this thesis provides unique insight into the properties of WT and familial mutant α-synuclein and their interaction with six PD-linked gene orthologues in budding yeast. WT and familial mutant α-synuclein was characterized in eight different yeast strains: one with no gene deletions, one with an established known moderator of α-synuclein deleted, three with orthologues of human PD-causing loss-of-function genes deleted and three with orthologues of human PD-risk genes deleted. Each gene deletion had different effects on WT and familial mutant α-synuclein’s properties. Certain deletions having broad effects on toxicity and localization, while others had familial mutant specific effects, and while still others had no effect.

 

One protein, Many Mutations

Each individual case of PD α-synuclein misfolding, accumulation, toxicity is present. However, how this becomes toxic is different for every case. In familial cases of PD, one particular gene that interacts with α-synuclein becomes mutated and gains a new toxic function or loses a healthy normal function, resulting in α-synuclein becoming toxic. Some of these mutations result in toxicity by impairing protein trafficking and causing α-synuclein to accumulate, others cause a loss in the ability to deal with cellular stress from α-synuclein and result cascading oxidative damage, while other mutations can be on α-synuclein and cause it change how it interacts and functions in cells. While these individual mutations are generally rare, they can each provide unique information into how α-synuclein functions. While many of these mutations have already been extensively analyzed, it is important to continue to research as many of the interactions between genes have yet to be explored. At the moment, at least 11 disease-causing genes have been identified and the list of PD risk genes continues to grow and is currently at forty-one with the most recent genome-wide association study (Chang et al., 2017). 

The Importance Multiple Model Systems

To truly understand how each of these genes and functions contributes to PD, it is important to examine them in multiple model systems. Each model system has its own advantages and disadvantages and models it’s intended disease in a distinct way. By using multiple model systems, the full complexity of diseases like PD can be understood. While the massive difference between yeast and humans may appear to be a drawback, it may  be one of its strengths. As seen in this study, yeasts allow for some of the quickest and clearest modeling of genetic interactions in living systems. However, as can be seen in the A30P and G51D α-synuclein mutants, properties in yeast do not always translate to humans so.

 

Parkinson’s Disease: Hope in Sight

As the world’s population continues to age, it will become more and more important to focus on diseases of age such as PD. PD strikes close to home and can affect people that we love and care for. While generally not fatal, PD does last for years and greatly reduces the quality of life for those suffering from it, their family, and their caregivers. With continued research into the mechanisms of proteins like α-synuclein, there will be a day where there is no longer PD.

 

 

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