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Eukaryon

Impact of Parkinson’s Disease Modifications on α-Synuclein Toxicity in a Yeast Model: SUMOylation and Phosphorylation

Rosemary Thomas
Lake Forest College
Lake Forest, Illinois 60045

Prelude: Our Amazing Brain

 

While writing this thesis, I could not stop thinking about a nun very dear to my heart. I knew Sister Sharron Hamm since I was only 13 years old. She lived alone in a small apartment room and loved having my family visit her. She would share her mouth-watering zucchini bread that she usually made too much of and saved in the freezer. For many years, I ate her zucchini bread and spent time with her on Sundays. I don’t remember when it started, but I always noticed her hands shaking uncontrollably. She had Parkinson’s disease. Eventually, once I got into Lake Forest College, it got so bad that she had to move into an elderly home an hour away from us. My family and I would go see her every once a month on weekends and always invited her to come and visit the church again for dinner parties, but she would say no, as she was too embarrassed of her shaking. At the end of our visits, we would lay out easy-to-peel cutie mandarins on her table, say our good-byes, and leave her room. A chilling fear would fill me every time I walked out the doors of the elderly home. Will she still be there waiting for us the next time? 

On November 19th, 2017, I got the news of her passing while doing the first trial of my SUMOylation temperature sensitive spotting. It was the most clear and compelling results I had in the spotting assay, about which you will read later. Now, every time I start a new spotting trial, I look at her picture in my lab drawer. My family was able to meet Sr. Sharon, visit her, talk to her, cry about her, and remember her because of our amazing brain. We are thankful for the brain and all the problems and challenges it brings, such as Parkinson’s disease, because without it, we would not have been as close to Sister Sharon as we were. Thank you, Sister Sharon’s brain.

 

Introduction

 

The Complex Brain

The brain is the most complex organ in the body, and it appears to be an impossible feat to decipher all 86 billion neurons communicating to control our thoughts, feelings, and actions. It is understandable, therefore, that the mechanisms underlying diseases of the brain are just as intricate. But this complexity and mystery serves to motivate neuroscience students and researchers, like myself, to understand the brain and its diseases more. 

One group of complex brain diseases are neurodegenerative disorders such as Alzheimer’s disease, Huntington’s disease, multiple sclerosis, prion diseases, and synucleinopathies (Figure 1a). Here, neurons in distinct regions of the brain degenerate, and misfolded proteins accumulate. For example, in Alzheimer’s disease, cells die in the cerebral cortex and hippocampus with the aggregation of beta amyloid and tau protein (Khachaturian, 1985; Masters et al., 1985). In Huntington’s disease, neurons die in the striatum with the aggregation of huntingtin protein (Lange, Thörner, Hopf, & Schröder, 1976; DiFiglia et al., 1997). In the case of synucleinopathies, the protein α-synuclein comes out of solution and aggregates in different regions of the brain (Figure 1b). Some examples of synucleinopathies include dementia with Lewy bodies, multiple system atrophy, Lewy body dysphagia, and Parkinson’s disease. In these disorders, protein misfolding and its connection to neuronal death is a central mystery. One contributing factor to misfolding could be due to the role of chemical modifications on these proteins. 

Proteins are modified after they are made. First, all proteins are encoded by our genes by which DNA is transcribed into RNA, and then RNA is translated into protein (Figure 2a). Then, this protein undergoes structural steps of protein folding to acquire a 3-D shape and is sent to its correct cellular location. Finally, these proteins are flexibly regulated a.  Neurodegenerative disorders 

 

  1. Synucleinopathies 

 

Figure 1. Neurodegenerative diseases

(a) In neurodegenerative diseases, neurons are dying within different regions of the brain. There is also the accumulation of misfolded protein. In Alzheimer’s disease, memory cells die with the accumulation of beta amyloid and tau tangles. In Huntington’s disease, prion diseases, and synucleinopathies, cells important for different types of movement die with the accumulation of huntintin protein, prion protein, or α-synuclein protein, respectively. 

(b) In synucleinopathies, α-synuclein accumulates in different regions of the brain. This thesis focuses on Parkinson’s disease in which α-synuclein first accumulates in the substantia nigra. 

through multiple posttranslational modifications to stabilize the folding and function of the protein (Figure 2b). Some of these modifications include a covalent and enzymatic addition of another molecule to an existing protein. In the case of neurodegenerative diseases, certain modifications lead to unstable protein folding and aid in the development of the disease. In other cases, the modifications could help counter the pathogenesis. More research must be performed to understand the roles of certain modifications in these diseases.

One modification is ubiquitination, in which a small ubiquitin protein covalently attaches to a target protein to tag it for later degradation (Glickman & Ciechanover, 2002). Ubiquitination has many other functions such as altering protein location within a cell, controlling protein activity, and allowing or preventing protein-protein binding (Mukhopadhyay & Riezman, 2007). In the case of neurodegenerative diseases, higher levels of ubiquitin are found in diseased neural tissue, and it is implicated as a major factor in the pathogenesis of various neurological diseases such as Alzheimer’s, Huntington’s, and prion diseases (Li et al., 1997 Ciechanover & Brundin, 2003). 

Similar to ubiquitin, but less well-studied, is a protein called Small Ubiquitin-like Modifier (SUMO) which covalently attaches to other proteins and modifies its function (Hay, 2005). In this case, the SUMO modification, or SUMOylation, does not tag proteins for degradation, but it does modify cellular processes, functions in cellular stress responses, and allows for protein solubility (Figure 3). Recently, SUMOylation was implicated in neurological diseases, as it was found in diseased neural tissue and protein aggregates within polyglutamine neurodegenerative diseases (Ross & Poirier, 2004). Even more recently, research demonstrates that the SUMO protein binds to the α-synuclein in the halos of Lewy bodies in Parkinson’s disease brains (Kim et al., 2011). My thesis will mainly focus on the role of SUMOylation in Parkinson’s Disease.

Thomas Fig 1

  1. Central dogma of biology

 

Thomas Fig 2

 

b. Modifications of protein

 

Figure 2. Creation of protein

(a) DNA is transcribed into RNA, and then RNA is translated into protein . This protein is then moved to its correct location within the cell.

(b) The protein is modified with a covalent attachment of a molecule. Some of these molecules lead to an unstable folding of the protein and might attribute to pathology. Other modifications can stabalize the shape of the protein. Some modifications include ubiquitination, nitration, glycation, phosphorylation, and SUMOylation. 

 

Thomas Fig 3

 

 

 

 

Figure 3. The role of SUMO

The modification of SUMOylation was discovered in 1996 and was similar to ubiquitination in the SUMO protein’s structure and ability to conjugate and deconjugate.  In this case, the SUMO protein can modify other proteins through covalent binding, regulate different SUMOylation enzymes, and modulate with subcellular localization, proteasomal function, and protein solubility (Eckermann, 2013).

Parkinson’s Disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease. In 2015 alone, it affected 6.2 million people (Dorsey et al., 2007; Serulle et al., 2006; Vos et al., 2016). PD usually onsets in patients after the age of sixty and leads to a progressive loss of movement control. Specifically, patients show symptoms of bradykinesia (slowness of movement), resting tremors, and muscle rigidity (Hughes, Daniel, Blankson & Lees, 1993; Olanow & Tatton, 1999). These symptoms usually present themselves after about ninety percent of the dopaminergic neurons in the substantia nigra pars compacta (SNc) dies (Squire et al., 2012; Figure 4a).  The SNc is part of the basal ganglia circuit, which allows for desired movements and represses undesired movements by the utilization of the neurotransmitter dopamine (Rabey & Hefti, 1990). This neuronal death in the SNc reduces the dopamine available to the basal ganglia, which eventually leads to the loss of movement control and the gain of tremors at rest commonly seen in patients. A closer look at the SNc with a microscope shows the visible formation of aggregates of misfolded protein called Lewy Bodies (Figure 4a). One of the main components of Lewy bodies is the misfolded protein α-synuclein, a suspected culprit of PD (Spillantini et al., 1997).

 

Two types of PD: Sporadic and familial

 

The cause of PD neuronal death in the SNc needs to be further explored. The most recent findings on the possible causes of PD are classified into two main groups: sporadic and familial causes (Goedert, 2001).

 

Thomas Fig 4

 

Sporadic PD makes up ninety percent of cases, where environmental and cellular stresses are possible major contributors to the disease. Some example factors that increase the risk of developing Parkinson’s disease include certain pesticides (Ascherio et al., 2006),

 

  1. Pathology of PD
  2. Familial and sporadic PD

 

Thomas Fig 5

 

Figure 4. Characterization of Parkinson’s disease

(a) In all cases of PD, researchers observe the misfolding of α-synuclein protein, its accumulation, and eventual aggregation.  Lewy bodies, one common pathology we see in PD, are mainly composed of these aggregated α-synuclein. It is still unknown how the aggregation of α-synuclein is associated to neuron death within PD. 

(b) In PD patients, like Sr. Sharron, more than 90% of dopaminergic cells have died in the substantia nigra even before the first symptoms of PD. When taking a closer look at the region where cells are dying, we can also see an accumulation of different protein called Lewy bodies. These Lewy bodies are mainly composed of misfolded α-synuclein protein. 

infections (Dobbs, Dobbs, Weller, & Charlett, 2000), heavy metal contact (Calne, Chu, Huang, Lu, & Olanow, 1994), oxidative stress (Langston, Langston, & Irwin, 1984), mitochondrial stress (Jenner & Olanow, 1996), and risk factor genes (Satake et al., 2009). 

Familial early-onset PD is caused by heritable mutations of genes and makes up ten percent of all PD cases. Main genes of interest include SNCA (Polymeropoulos et al., 1997), UCHL1 (Leroy et al. 1998), Parkin (Lücking et al., 2000), LRRK2 (Funayama et al., 2002), DJ-1 (Bonifati et al., 2003), PINK1 (Valente et al.,2004), and ATP13A2 (Ramirez et al., 2006). UCHL1 and Parkin mark protein for proteasome degradation (Shimura et al., 2000; Leroy et al., 1998). Mutations in these genes lead to a buildup of proteins that are toxic to the cell.  LRRK2 usually codes for proteins that assist in intracellular signaling and kinase activity (Dachsel et al., 2007). LRRK2 mutation leads to excessive kinase activity that is toxic to the cell (Greggio et al., 2006). DJ-1, PINK1, and Parkin are key players in proper mitochondria functioning and impairment leads to mitochondrial damage and oxidative stress (Norris & Giasson, 2005; Deng, Le, Shahed, Xie, & Jankovic., 2008; Yang et al., 2005). There are more PD genes that still need to be discovered. 

One of the most well-studied genes linked with PD is the SNCA gene which codes for the α-synuclein protein. In this case, duplication and triplications of the SNCA gene leads to PD (Chartier-Harlin et al., 2004). This demonstrates how overexpression of α-synuclein is linked with toxicity. Whether the type of PD is sporadic or familial, all cases of PD exhibit a misfolding and accumulation of the α-synuclein protein within the brain (Pollanen, Dickson, & Bergeron, 1993; Caughey & Lansbury, 2003; Figure 4b). Further research must be performed to understand what the role of α-synuclein is in PD and why it accumulates. In this thesis, my research focuses on α-synuclein.

 

α-Synuclein 

α-Synuclein is a protein encoded by the SNCA gene, and it accumulates in all cases of Parkinson’s disease. It is 140 amino acids long and split into three domains: The N-, NAC, and C- domains. This protein is found in the tips of neurons called presynaptic terminals, in the cytoplasm, and found bound to membrane fatty acids (Maroteaux & Scheller, 1991). More specifically, α-synuclein is in the presynaptic terminals of neurons that release dopamine, the neurotransmitter needed for proper movement control and inhibition of unnecessary movement. Previous research explains that α-synuclein might function to mobilize the synaptic vesicles that carry and release dopamine (Cabin et al., 2002, Burré et al., 2010). α-Synuclein also plays a role in vesicle trafficking from the endoplasmic reticulum (ER) to the Golgi Apparatus (Cooper et al., 2006). In rodents, α-synuclein deletion does not lead to any significant phenotypic changes, but α-synuclein deletion, in addition to deletion of similar proteins β-synuclein and ɣ-synuclein, leads to an age-dependent diminishment in transmission and excitatory synapse size (Chandra et al., 2004, Greten-Harrison et al., 2010).

In the brain of PD patients, α-synuclein somehow gains toxicity, as it misfolds and disrupts many aspects of normal cell functioning. The misfolded protein starts to aggregate and accumulate in the ER-Golgi pathway to block the transport of newly made protein (Cooper et al., 2006). It eventually inhibits the proteasome and autophagy pathways, the main systems that degrade unneeded and damaged proteins. This inhibition gives rise to further protein accumulation (Martinez-Vicente et al., 2008).  α-Synuclein also induces tau fibrilization that later disrupts the stability of microtubules (Giasson et al., 2003).  

α-Synuclein dysfunction clearly relates to the development of PD, but why α-synuclein becomes toxic and how it associates with neuron death is also still unclear. As stated before, aggregates of misfolded protein, Lewy bodies (LBs), are mainly composed of the misfolded and aggregated α-synuclein protein (Spillantini et al., 1997). Whether formation of LBs promotes pathology or protection is still debated on, but recent studies suggest that it might be protective (Braak, Sastre, Bohl, de Vos, & Del Tredici, 2007; Auluck, Caraveo & Lindquist, 2010).  The main form of PD toxicity might stem from a protofibril intermediate of α-synuclein that precedes LB development (Caughey & Lansbury, 2003). LBs might develop to form stable fibrils that sequester toxic protofibril intermediates of α-synuclein as a protective mechanism. Unfortunately, LB formation might start to extend beyond the SNc and lead to more widespread neurodegeneration (Volpicelli-Daley et al., 2011).

 

Familial mutations

Six familial point mutations of the α-synuclein gene lead to early onset Parkinson’s disease. The three older familial mutations are A53T, A30P, and E46K. The A53T α-synuclein mutant was first discovered in a Greek family in 1997 and is not toxic in budding yeast compared to wildtype (WT), but does form aggregates (Polymeropoukous et al., 1997; Sharma et al., 2006; Brandis et al., 2006). The A30P mutant was found in German families in 1998 and discovered, through in vitro and in vivo studies, to not bind to cell membranes and remain cytoplasmically diffuse throughout the cell (Krüger et al., 1998; Jensen, Nielsen, Jakes, Dotti, & Goedert, 1998; Sharma et al., 2006; Brandis et al., 2006). The E46K mutant was first found in a Spanish family in 2004 and binds to lipids in budding yeast but increases aggregation in neuroblastoma cells (Zarranz et al., 2004; Fiske et al., 2011). Three familial mutations were discovered recently in 2013 and onwards and, therefore, need more research. The H50Q α-synuclein mutant was first discovered in 2013 in an English patient and leads to more in vitro and in vivo aggregation and toxicity in budding yeast (Proukakis et al. 2013; Rutherford, Moore, Golde, & Giasson, 2014; Fares et al., 2014). The G51D mutant was found in a British family in 2013 and does not display membrane binding or aggregates, but instead stays diffuse throughout the cytoplasm similar to the A30P mutant in budding yeast (Kiely et al., 2013; Fares et al., 2014). A53E was the last familial mutant discovered. This α-synuclein mutation was discovered in a Dutch family in 2014 and is suggested to lead to less in vitro aggregation and membrane binding (Pasanen et al., 2014; Ghosh et al., 2014). In budding yeast, A53E exhibits less membrane binding, but has similar toxicity to WT (Tembo, Thesis, 2014).

 

Modifications of α-synuclein

α-Synuclein is also heavily modified by other proteins and functional groups. It can undergo ubiquitination (Shimura et al., 2001), nitration (Clayton & George, 1998), phosphorylation (Okochi et al., 2000; Fujiwara et al., 2002), and SUMOylation (Dorval & Fraser, 2006; Krumova et al., 2011). In PD, the ubiquitin-proteasomal pathway is disrupted; this disruption leads to the abnormal accumulation of ubiquitinated α-synuclein in LBs (Shimura et al., 2001; Chung, Dawson, & Dawson, 2001). Nitration of α-synuclein increases fibril formation and aggregation (Hodara et al., 2004; Burai, Ait-Bouziad, Chiki, & Lashuel, 2015). Nitration, specifically at T39 of α-synuclein, reduces α-synuclein membrane binding. This thesis will focus on the other two modifications mentioned: SUMOylation and phosphorylation. Both SUMOylation and phosphorylation are found on the α-synuclein within LBs of PD patients. 

Phosphorylation is the covalent attachment of a phosphoryl group to another protein, in this case, α-synuclein. It modifies protein conformation to regulate enzyme activity (Alberts et al., 2013). Accumulated α-synuclein in LBs are highly phosphorylated at two serine sites of S129 and S87 (Fujiwara et al., 2002; Paleologou et al., 2010). In PD patients, about 90% of the α-synuclein within LBs are phosphorylated at S129 verses less than 4% of α-synuclein in healthy patients’ brains (Sato, Kato, & Arawaka, 2013). The role of phosphorylation of α-synuclein in PD has conflicting evidence. One lab used a Drosophila model to show that a mutation of serine (S) to an alanine (A) to block phosphorylation (S129A) on α-synuclein decreased toxicity, while a mutation of serine (S) to an aspartic acid (D) to mimic phosphorylation (S129D) increased α-synuclein toxicity (Chen & Feany, 2005). Another lab used a rat model and found conflicting results to the Drosophila study. The S129A mutation to block phosphorylation increased nigrostriatal degeneration, while the S129D mutation to mimic phosphorylation led to no changes in nigrostriatal pathology (Gorbatyuk et al., 2008). Another lab found similar results where the S129A mutant increased toxicity in rats (Azeredo da Silveira et al., 2009). Contrastingly, a paper published in the same year observed no difference in toxicity of the WT, S129A, and S129D α-synuclein mutants in rats (McFarland et al., 2009). In budding yeast, the S129D phosphorylation mimic mutation decreased plasma membrane association (Fiske et al., 2011). 

The other modification, SUMOylation, modulates protein-protein binding and influences subcellular localization of α-synuclein (Dohmen , 2004). Although there is new identification of SUMO targets, the function of SUMO on many of those targets remains obscure and could be different depending on the target. One target of the SUMO protein is α-synuclein, in which the SUMO protein forms a covalent bond with α-synuclein. SUMOylation occurs at two main sites of α-synuclein: K96 and K102 (K96 and K102 refers to the Lysine (K) at the 96th or 102nd amino acid position). In vitro and cell studies demonstrate that mutations of the lysine (K) to arginine (R) blocks SUMOylation at those α-synuclein sites (Krumova et al., 2011). These cell studies reveal an increase of aggregation and cytotoxicity of α-synuclein when SUMOylation was impaired at both sites of α-synuclein. This demonstrates that SUMOylation possibly promote solubility of aggregation-prone protein like α-synuclein. 

 

PD Model Organism: Yeast

Various models are utilized to study PD: cell cultures, C. elegans, Drosophila, rats, and mice (Nass & Prezdborski, 2008). The Dr. DebBurman lab uses the model organism of yeast. The entire genome for these yeasts are sequenced, and they have high homology with human genes. These yeasts make, fold, and degrade protein like humans to allow for correlation of findings in yeast with the role of alpha-synuclein in humans. In addition, since the yeast genome is fully sequenced, it is easy to perform genetic manipulations (Outiero & Lindquist, 2003; Willingham, Outeiro, DeVit, & Muchowski, 2003; Dixon, Mathias, Zweig, Davis, & Gross, 2005; Zabrocki et al., 2005; Sharma et al., 2006; Brandis et al., 2006; Nass & Prezdborski, 2008). Yeast grow fast, have a short lifespan, are cheap, and easy to handle (Nass and Prezdborski, 2008). These traits make them an excellent model organism to use in undergraduate research.

Outside of PD, yeast can successfully model various human diseases such as cancer, Alzheimer’s disease, and Huntington’s disease (Nass and Prezdborski, 2008; Komano et al., 1998; Meriin et al., 2002). Yeast might not exactly model a neuron in human brains, but it is a practical and excellent alternative to PD research that gives insight into the toxicity, accumulation, and localization of α-synuclein (Outiero & 

Lindquist, 2003; Brandis et al., 2006; Sharma et al., 2006). Research performed with yeast recently got a Nobel prize in 2016 making a grand total of five Nobel prizes awarded for work done in yeast.

In my research, I used two yeast models: budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe). These yeasts do not have the α-synuclein gene. Therefore, our lab transforms a plasmid vector (a circular piece of DNA) that contains the α-synuclein gene into yeast (Figure 5). In the Lindquist lab, the addition of a single copy of the α-synuclein gene did not cause toxicity in budding yeast, but two copies led to cell death and aggregate formation similar to LBs (Outiero & Lindquist, 2003). In budding yeast, it is typical to see α-synuclein bound to the cell membranes (Sharma et al., 2006). In fission yeast, α-synuclein is mainly aggregated within the cell and does not easily bind to the membrane (Brandis et al., 2006). 

The DebBurman lab has previously used budding yeast and fission yeast to study the familial mutations of α-synuclein (Sharma et al., 2006; Brandis et al., 2006; Tembo, Thesis 2014; Ong, Thesis, 2017), modifications of α-synuclein (Fiske et al., 2011), PD risk factor genes (Jones, Thesis, 2018), and more. Sharma et al., (2006) analyzed WT α-synuclein and the A53T mutant in budding yeast. They found no difference in toxicity between WT and A53T mutant α-synuclein. Brandis et al., (2006) assessed A53T α-synuclein in fission yeast and found it to be toxic. Senior thesis student, Wase Tembo, characterized the three new familial mutants (H50Q, G51D, and A53E) in both budding yeast and fission yeast. In budding yeast and fission yeast, the H50Q and A53E mutant exhibited toxicity, while the G51D mutant had no change in toxicity from WT α-synuclein (Tembo, Thesis, 2014). Fiske et al., (2011) used both budding yeast and fission yeast to assess phosphorylation of α-synuclein and found less membrane binding when phosphorylation was mimicked in budding yeast. Both blocking and mimicking phosphorylation increased membrane binding in fission yeast. Another senior thesis student, Paul Jones, combined loss-of-function PD genes with the familial mutants of α-synuclein (Jones, Thesis, 2018). He used the risk genes to give insight into each familial mutant’s mechanism of toxicity in budding yeast. 

 

Figure 5. Budding yeast and fission yeast

In the following experiments, plasmids containing the α-synuclein gene were transformed into budding yeast (left) and fission yeast (right). This plasmid contains a galactose inducible promoter for budding yeast and a thiamine repressible promoter for fission yeast. Both types of yeast share many genes with humans. Yeast also make, fold, and degrade protein just like humans. In addition, both budding yeast and fission yeast can exhibit the pathological properties of α-synuclein: membrane binding and aggregation. These abilities make yeast an excellent model organism to study the effects of α-synuclein protein. 

 

Gap in Knowledge

Previous researchers studied SUMOylation of α-synuclein in vitro and in cell lines (Krumova et al., 2011), but SUMOylation has not been well studied in a living model organism such as yeast. In addition, aggregated α-synuclein is heavily modified by multiple modifications at the same time. Little is known on the effect of different modifications on α-synuclein’s folding. Specifically, the effect of combining SUMOylation and phosphorylation with the familial mutants of α-synuclein is unknown.  More findings on the relationship between SUMOylation and phosphorylation could also yield more information on the role of phosphorylation of α-synuclein itself.

 

Hypothesis

I hypothesized that SUMOylation is protective and will decrease the aggregation and toxicity of α-synuclein. Based on this hypothesis, I proposed that sumoylation will counteract phosphorylation toxicity and regulate the toxicity of the familial mutations of α-synuclein. To test these hypotheses, I had five aims summarized below and in Figure 6. 

 

My Study

Chapter 1: Blocking SUMOylation on α-synuclein

Aim: To assess the effect of SUMOylation on α-synuclein toxicity and aggregation, I made mutations to block SUMOylation (K96R, K102R, K96R/K102R) in budding yeast.

Main Findings: When SUMOylation was blocked in budding yeast, K96R and K96R/K102R mutants show increased toxicity. K102R demonstrate no significant change in the toxicity. Blocked SUMOylation leads to increased α-synuclein aggregation. 

 

Chapter 2: Altering levels of SUMOylation in yeast

Aim: To further understand the effect of SUMOylation on α-synuclein, I used genetically modified temperature sensitive yeast strains that had excess (ulp1ts) and deficient (smt3ts) levels of SUMO. I specifically evaluated the impact of excess and deficient levels of SUMOylation on the toxicity and localization of the three newer familial mutants of α-synuclein (H50Q, G51D, A53E). 

Main Findings: Excess levels of SUMO leads to more survival of budding yeast compared to deficient levels of SUMO. Deficient levels of SUMO lead to more toxicity, in which even G51D, a familial mutant that usually does not show toxicity, grew considerably less. 

 

Chapter 3: Examine SUMOylation and phosphorylation

Aim: To evaluate the how one modification affects another modification’s properties, I observed the effects of blocking or mimicking phosphorylation in addition to blocking SUMOylation on α-synuclein.

Main Findings: Blocking both phosphorylation and SUMOylation on α-synuclein leads to better survival than the SUMOylation-deficient mutants alone. Phosphorylation mimics and blocking of SUMOylation on α-synuclein together leads to similar levels of toxicity than SUMOylation-deficient mutants alone. The blocking of both modifications leads to more membrane binding, while phosphorylation mimic with SUMOylation blocking leads to cytoplasmic diffusion. 

 

Chapter 4: SUMOylation impacts the toxicity of familial mutants

Aim: To evaluate the impact of modifications on the toxicity and localization of the familial mutations, I blocked SUMOylation and either mimicked or blocked phosphorylation in combination with the familial mutations.

Main Findings: The blocking of SUMOylation leads to A53T α-synuclein toxicity. Blocking both SUMOylation and phosphorylation leads to better growth for E46K α-synuclein. Blocking SUMOylation and mimicking phosphorylation leads to less growth for A53T α-synuclein. The localization patterns of the familial mutations dominate over the localization patterns when either SUMOylation is blocked alone or when both SUMOylation and phosphorylation are blocked. Phosphorylation mimics with the blocking of SUMOylation leads to cytoplasmic diffusion. 

 

Chapter 5: Is SUMOylation always protective?

Aims: To further assess the effect of SUMOylation on α-synuclein, I made mutations to block SUMOylation (K96R, K102R, K96R/K102R) in the model organism of fission yeast. 

Main Findings: Blocked SUMOylation in fission yeast leads to decreased toxicity and smaller aggregations compared to WT α-synuclein.

 

Figure 6. Aims of SUMOylation project

(a) SUMOylation is thought to be protective in vitro, therefore, I predict blocking SUMOylation will lead to more aggregation and toxicity in budding yeast (Ch. 1) and fission yeast (Ch.2). 

(b) When placing α-synuclein in a yeast strain with excess SUMO (ulp1ts), I predict there to be less toxicity and aggregation. When placing α-synuclein in a yeast strain with deficient SUMO levels (smt3ts), I predict more toxicity and aggregation in budding yeast.

(c) It is possible that phosphorylation might lead to dysfunction in normal α-synuclein. When blocking SUMOylation and phosphorylation, I predict there to be less aggregation and toxicity because there is a reduction in phosphorylation. When SUMOylation is blocked and phosphorylation is mimicked, I predict more toxicity and aggregation.

(d) The familial mutants lead to early onset PD and is predicted to cause increased toxicity and aggregation when combined with SUMOylation blocking mutations and phosphorylation. 

 

MATERIALS AND METHODS

All methods were based on methods from Outiero and Lindquist (2003), Brandis et al. (2006), Sharma et al. (2006), Fiske et al. (2011), and Tembo, Thesis, 2014.

 

Creation of α-synuclein Constructs

In chapter 1, I created K96R, K102R, and K96R/K102R mutants to block SUMOylation on the single sites (K96 and K102) or on both sites at the same time (K96/K102) to understand the effects of SUMOylation on α-synuclein. In chapter 2, the H50Q, G51D, and A53E mutant α-synuclein were transformed into temperature sensitive yeast strains, ulp1ts and smt3ts, to understand the effects of excess and deficient levels of SUMOylation. In chapter 3, I created phosphorylation blocking mutations (S129A or S87A/S129A) and phosphorylation mimicking mutations (S129D or S87D/S129D) on an α-synuclein background that already had SUMOylation blocked (K96R/K102R) to understand the effect of phosphorylation on SUMOylation’s effects. In chapter 4, I created SUMOylation blocking mutation (K96R/K102R) and phosphorylation altering mutants with the 6 familial mutations (A30P, E46K, A53T, H50Q, G51D, and A53E) to understand the how SUMOylation effects the familial mutations.  In chapter 5, I placed the SUMOylation blocking mutations made for chapter 1 into the fission yeast model organism.

 

Yeast Stains

Experiments conducted used budding and fission yeast model organisms. The budding yeast strain was BY4741 and contained the pYES1.1 vector that carried α-synuclein tagged with enhanced green florescent protein (eGFP) (Table 1). The fission yeast strain was TCP1 and contained the pNMT1 vector that has α-synuclein tagged with green florescent protein (GFP). Two additional budding yeast strains were used: ulp1tsts and smt3ts. Ulp1ts has a defect in the gene for the ub1-specific protease that deconjugates the SUMO protein from substrates at 30 degrees (Li, Liu, Chen, Kharbanda, & Kufe, 2003; Shahpasandzadeh et al., 2014). Smt3ts has a defect in the smt3-331 gene that codes for the yeast SUMO homologue, smt3, at 30 degrees (Shahpasandzadeh et al., 2014). 

 

Site-directed Mutagenesis

We created all mutations (the SUMOylation blocking, phosphorylation blocking, phosphorylation mimicking, and familial mutations) using the Invitrogen site-directed mutagenesis kit. I followed the GENEART Site-Directed Mutagenesis System Protocol by Invitrogen Life Technologies. Table 2 shows the specific primers used for each chapter’s mutations. The mutant DNA was then transformed into (taken up by) One Shot Max Efficiency Dh5α-T1 component cells (E. coli bacteria cells) that were grown on LB+Ampicillin agar plates. Plasmid DNA was isolated from the bacteria using a Qiagen Mini-prep kit. The DNA was then sent to the University of Chicago DNA Sequencing Facility to be sequence confirmed. Then, the DNA was transformed into yeast (Figure 7). 

 

Yeast Expression

The pYES1.1 vector within budding yeast has a GAL promotor in which α-synuclein will only be expressed in the presence of galactose in the media. If the yeast is grown in glucose, α-synuclein is repressed. The pYES1.1 vector also has a gene to produce uracil. The budding yeast cells were grown in media with no uracil (SC-Ura) that acts as a selection pressure, so only plasmid that has the uracil gene will grow on this media. The pNMT1 vector of fission yeast express and repress α-synuclein depending on whether thiamine is present in Edinburg Minimal Media (EMM). Fission yeast express high levels of α-synuclein in thiamine absence (EMM-T) and express low levels of α-synuclein in thiamine presence (EMM+T). 

 

GFP Fluorescent Microscopy: To Observe α-synuclein Localization within the Cells

Yeast cells were grown in 3mL of SC-Ura glucose (budding yeast) or EMM+T (fission yeast) overnight in the 30°C shaking incubator at 200rpm. In the case of the temperature sensitive strains (ulp1ts and smt3ts), the cells were grown in 25°C. After, the cells were washed three times with 5mL of water while using a centrifuge to pellet the cells at 15000Xg. The cells were transferred to SC-Ura galactose (budding yeast) or EMM-T (fission yeast) and expressed at 30°C. In the case of the ulp1ts and smt3ts strains, one set of cells were put in 30°C (genes of strains are defective) and in 25°C (no defect in genes). 1mL of cells was removed from the media and pelleted after 6, 12, 18, and 24 hours (12, 24,36, and 48 hours for fission yeast). After, 10uL of the pelleted cells and some liquid was pipetted onto a glass slide and observed under a TE2000-U Nikon fluorescent microscope at 1000X. As stated before, all α-synuclein is tagged with a GFP. Because of this, our α-synuclein turns green when placed under the blue light of this florescent microscope. Next, images of approximately 1000 cells were taken at each timepoint for each sample and quantified using Metamorph 4.0 software. This procedure is repeated until there are around 6 trials. The cells are classified according to different established phenotypes shown.

Budding yeast:

 

Statistics

The quantifications from the live cell images were represented in a graph showing the percentage of cells that display one of the three dominant localization patterns (diffuse, halo, aggregation/foci) for each mutant across the time points. Next, a Chi-square for goodness of fit was performed to evaluate if the mutants were significantly different from the expected localization distribution. In chapter 1, WT was used as the expected localization distribution. In chapter 2, the mutant at 25°C (normal levels of SUMOylation) was used as the expected localization distribution. In chapter 3, K96R/K102R was used as the expected localization distribution. Error bars in bar graphs represent the percentage of error of one sample to the next. To measure the consistency of scoring for localization patterns, a reliability test was performed with Paul Jones, a researcher in the Dr. DebBurman lab. We got a reliability percentage of 85%.  

 

Spotting Assay: To Test Toxicity and Cell Growth

Yeast cells were grown in 3mL of SC-Ura glucose (budding yeast) or EMM+T (fission yeast) overnight in the 30°C shaking incubator at 200rpm. For the temperature sensitive strains (ulp1ts and smt3ts), the cells were grown in 25°C. After, the cells were washed three times with 5mL of water while using a centrifuge to pellet the cells at 15000Xg. 1mL of the cells are placed in a separate tube and counted using a hemocytometer. Once the cell density is calculated, 2.0X106 cells was removed and placed in enough water to make a 1mL suspension. After, cells were diluted 5-fold in a 96 well microtiter plate. Then, the cells were spotted onto SC-Ura glucose and SC-Ura galactose (budding yeast) or EMM-T and EMM+T (fission yeast) agar plates. The plates were grown in the 30°C incubator for two days and then imaged by an HP Canoscan scanner and Adobe Photoshop CS3. In the case of the temperature sensitive strains, (ulp1ts and smt3ts), there was also sets of plates grown in 25°C to analyze the effect of α-synuclein without the effect of the strains.

 

Western Blot Assay: To Observe How Much Protein is Expressed

Budding yeast cells were grown two days before in SC-URA glucose. The cells were pelleted the next day and washed with water three times. After, they were re-suspended in water and counted with a hemocytometer. The cells, now at a volume of 15X10^7 cells/mL, were washed with 100mM NaN3 and solubilized in electrophoresis sample buffer (ESB). These produced cell lysates that were run on a 10-20% Tris-Glycine gel (Invitrogen) at 130 volts with 1x SDS running buffer. SeeBlue ladder allowed for us to determine the size of the protein. The gels were then transferred to Polyvinylidene fluoride (PVDF) membranes using a semi-dry transfer technique. The α-synuclein antibody was used to probe the protein on the membrane. After, a secondary antibody was used. The control was the phosphoglycerokinase (PGK) antibody (Molecular Probes). 

 

Table 1. α-Synuclein controls and mutations

The table shows the different α-synuclein controls and mutations used in the experiments. The table also gives information on the plasmid, yeast strain, and fluorescent tags used. 

 

Table 2. Mutant primer design 

The table shows the primers used to make the α-synuclein mutations used in each of the experiments conducted. Each mutated codon is in the middle of the primer. 

 

Figure 7. Creation of mutations

Polymerase chain reaction and an Invitrogen site-directed mutagenesis kit were used to make the mutation. WT plasmid (pYES11 or pNMT1) was combined with enzymes that methylate, primers (small pieces of DNA that contain the mutation), and other solutions. The forward and reverse primers bound to the plasmid and recreated the template plasmid but now with the desired mutations. These mutated plasmids were transformed into E. coli where they are amplified. Then, we purify the plasmid and send it to the University of Chicago DNA sequencing facility to check for correct sequences. The corrected sequenced plasmid DNA is transformed into yeast. 

 

CHAPTER 1

BLOCKING SUMOYLATION ON α-SYNUCLEIN

 

Experimental Set-up

The first goal was to understand the effect of α-synuclein SUMOylation in budding yeast. Alex Roman ’16 and I started this project by creating the SUMOylation blocking mutations of α-synuclein. We mutated the lysine of K96 and K102 to an arginine (R) which has similar properties to lysine (a positive charge) but cannot get SUMOylated. Therefore, K96R and K102R mutations were made on α-synuclein to block SUMOylation. To assess the effects of blocked SUMOylation on α-synuclein, I asked three questions. First, I asked how toxic the mutants were to yeast by using a serial dilution spotting assay. Next, I asked where α-synuclein is located within the yeast by using fluorescence microscopy because α-synuclein is tagged with a green fluorescence molecule (GFP). Finally, I analyzed the amount of the α-synuclein made in yeast by using Western blots. 

 

Blocked sumoylation increases toxicity

In PD, cell death is associated with α-synuclein toxicity. This α-synuclein toxicity can be modeled in yeast by using the spotting assay. In this assay, yeast is serially-diluted five-fold and grown on media that can induce (SC-Ura galactose) and repress (SC-Ura glucose) α-synuclein expression. SC-Ura glucose plates, therefore, act as a control to check if I used the equal numbers of yeast cells in each lane before testing the samples. SC-Ura galactose plates express α-synuclein, so the effects of the SUMOylation blocking mutations can be assessed and compared to the growth of WT α-synuclein tagged with eGFP (WT-eGFP). I used two other controls: vector alone (no α-synuclein) and GFP (vector contains only GFP). 

I specifically assessed whether blocked SUMOylation (K96R, K102R, and K96R/K102R) of α-synuclein was toxic in budding yeast and hypothesized that blocked SUMOylation would lead to less cell growth. I found that K102R and K96R/K102R both had less cell growth compared to WT (Figure 8a). K102R had similar levels of growth to WT α-synuclein. Uniform growth on glucose plates indicated that growth differences were specifically due to α-synuclein and its variants.

 

Blocked SUMOylation increases aggregation and reduces membrane association

In a PD brain, α-synuclein has altered cellular localization, often more aggregation and membrane association. I asked if α-synuclein localization would be altered in similar ways when I blocked SUMOylation in yeast. Using live-cell fluorescence microscopy, I observed GFP-tagged α-synuclein localization in yeast over 24 hours of . First, I observed WT α-synuclein. Over this time course, WT α-synuclein first appeared to associate with intracellular foci (6 hr) and then re-localized to the plasma membrane by 12 hours (Figure 9a). In contrast, all three SUMOylation blocking mutants (K96R, K102R, and K96R/K102R) exhibited more aggregation early and persisted into the time course. 

To quantitatively assess the qualitative observations I initially found, I repeated these microscopy trials 5 times and then scored the α-synuclein localization for five distinct patterns (diffuse, diffuse/halo, halo, foci, foci/halo) (Halo refers to α-synuclein bound to the plasma membrane).  I have displayed graphs that present the same data in different ways. In figure 10, I graphed each timepoint with the mutations and the percentage of cells for the five localization patterns. In figure 9b, I graphed the percentage of cells that display one of the three dominant localization patterns (diffuse, halo/plasma membrane, aggregation/foci) for each mutant across the time points. In chapters 2-5, I only the graphs similar to figure 9b with the three dominant localization patterns will be shown. I then performed a chi-square test for goodness of fit to evaluate if the mutants were different from WT a-synuclein in their localization distribution. The expected values of WT for the diffuse phenotype was 61%, 33%, 45%, and 24% for 6hrs, 12hrs, 18hrs, and 24hrs, respectively.  The expected values of WT for the plasma membrane phenotype was 69%, 73%, 84%, and 92% for 6hrs, 12hrs, 18hrs, and 24hrs, respectively. The expected values of WT for the aggregation phenotype was 12%, 31%, 13%, and 5% for 6hrs, 12hrs, 18hrs, and 24hrs, respectively. I noted a significant increase in aggregation for the SUMOylation blocking mutations in comparison to WT at 6, 12, and 18 hours (p=<.01; Figure 9b). The SUMOylation blocking mutations also had significantly fewer cells with membrane binding at 6 hours (p=<.05). Finally, yeast with the SUMOylation blocking mutations exhibited significantly less cytoplasmically diffuse α-synuclein at 18 hours (p=<.01). 

 

Amount of α-synuclein expressed is inconclusive

In PD, α-synuclein protein accumulates. To assess the change in the amount of α-synuclein after SUMOylation was blocked, I performed Western blots (Figure 8b). The anti-α-synuclein antibody was used to tag α-synuclein protein. Anti-PGK antibody was used as a control to make sure there amounts of yeast protein were loaded. After three trials, each Western blot was inconsistent in terms of the amount of α-synuclein expression and the thickness of the control bands. Another example of an inconsistent Western blots is shown in Appendix B (p. 95). 

a. Toxicity

 

 

b. Expression

 

Figure 8. Toxicity and protein expression of SUMOylation blocking mutations 

(a) Spotting assay with five-fold serial dilutions of yeast with Vector, GFP, WT-eGFP, K96R, K102R, and K96R/K102R in repressive (SC-Ura glucose) and inducive (SC-Ura galactose) media (n=6). 

(b) Western blot at 24 hours after α-synuclein expression for WT, K96R, K102R, and K96R/K102R. The anti-α-synuclein and anti-PGK loading control was used (n=3).

 

a. Localization

 

b. Localization Quantification

 

Figure 9. Microscopy and quantification of SUMOylation blocking mutations

(a) The microscopy images shown are of the dominant phenotype observed for WT, K96R, K102R, and K96R/K102R variants over 24hrs. The images were taken at 6, 12, 18, and 24 hours after induction in SC-Ura galactose (n=6). 

(b)The time course quantification for the dominant phenotypes shown by WT, K96R, K102R, and K96R/K102R variants in a bar graph (n=4).

 

CHAPTER 2

ALTERING LEVELS OF SUMOYLATION IN YEAST

Experimental Set-up

My second goal was to assess the impact of SUMOylation by using genetically modified yeast strains that had altered levels of SUMOylation. These strains were gifted to the DebBurman Lab by Mark Hochstrasser’s lab (Yale University). The first strain, ulp1ts, has a temperature sensitive defect at 30°C in the gene for the ub1-specific protease that cleaves the SUMO protein from substrates (Li, Liu, Chen, Kharbanda, & Kufe, 2003; Shahpasandzadeh et al., 2014). Another strain, smt3ts, has a temperature sensitive defect at 30°C in the smt3-331 gene that codes for the SUMO protein (Shahpasandzadeh et al., 2014). This allows for ulp1ts to give excess levels of SUMO at 30°C and smt3ts to give deficient levels of SUMO at 30°C. At 25°C, the genes are not defective and, therefore, have normal levels of SUMO. I evaluated WT and the three newer familial mutations of PD (H50Q, G51D, and A53E) in the two strains. 

 

H50Q, G51D, and A53E grow better in excess SUMO and are toxic in deficient SUMO

First, I observed the toxicity of excess and deficient levels of SUMO (Figure 11). There was sufficient uniform growth on the control repressive glucose media at 25°C with no effect of the stains. This equal growth in each lane accounted for even cell dilutions.

 In the galactose media at 25°C, I observed the effects of α-synuclein without the effects of the strains. In this condition, regardless of strain and consistent with previous research, the H50Q mutant had less cell growth than WT α-synuclein. G51D grew as well or slightly better than WT α-synuclein. A53E grew similarly to WT α-synuclein.  

In glucose media at 30°C, I observed the effects of the strain without the effects of α-synuclein. Here, WT α-synuclein and the familial mutants with excess SUMO had greater survival, while WT α-synuclein and the familial mutants with deficient SUMO grew less. 

In galactose media at 30°C, I observed both the effect of α-synuclein and the effect of the strains. WT, G51D, and A53E α-synuclein all grew better with excess SUMO. WT and all the mutants grew less with deficient SUMO compared to the mutants grown on galactose at 25°C where there was only the effect of α-synuclein and no effect of the strains.

 

H50Q, G51D, and A53E show no localization change with more SUMO

Next, I compared the change in localization of α-synuclein with excess SUMO protein (30°C) and normal levels of SUMO protein (25°C) in the ulp1ts strain. In figure 12, I did not observe a difference in the localization patterns for H50Q, G51D, or A53E between excess and normal SUMO levels. H50Q exhibited aggregation whether there were normal or excess levels of SUMO. G51D exhibited diffusion with normal or excess SUMO. A53E exhibited aggregation and diffusion with normal or excess SUMO. I then performed a chi-square test for goodness of fit to evaluate if there was a significant difference in localization pattern between the mutants with excess or normal levels of SUMO. As seen in figure 13, there was no significant difference. 

 

Statistics information on more SUMO levels

The expected values of H50Q at 25°C for the diffuse phenotype was 52%, 35%, and 42% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of H50Q at 25°C for the plasma membrane phenotype was 19%, 18%, and 36% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of H50Q at 25°C for the aggregation phenotype was 29%, 47%, and 22% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of G51D at 25°C for the diffuse phenotype was 99%, 97%, and 91% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of G51D at 25°C for the plasma membrane phenotype was 1%, 1%, and 8% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of G51D at 25°C for the aggregation phenotype was 0%, 2%, and 1% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of A53E at 25°C for the diffuse phenotype was 64%, 51%, and 42% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of A53E at 25°C for the plasma membrane phenotype was 19%, 33%, and 39% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of A53E at 25°C for the aggregation phenotype was 25%, 16%, and 19% for 12hrs, 18hrs, and 24hrs, respectively.

 

H50Q, G51D, and A53E show no localization change with deficient SUMO

Next, I compared the change in localization of α-synuclein with deficient levels of SUMO protein (30°C) to normal levels of SUMO protein (25°C) in the smt3ts strain. In figure 14, I did not observe a difference in the localization patterns for H50Q, G51D, or A53E. Similar to the results found with excess SUMO, H50Q exhibited aggregation whether there were normal or deficient levels of SUMOylation. G51D exhibited diffusion with normal or deficient levels of SUMOylation. A53E exhibited aggregation and diffusion with normal or deficient SUMOylation. I then performed a chi-square test for goodness of fit to evaluate if there was a significant difference in localization pattern in the living cells between the mutants with deficient or normal levels of SUMO. As seen in figure 15, I found no significant differences. One observation was the increase in dead, non-fluorescing cells, which were not included in the quantification.

 

Statistics information on deficient SUMO levels

The expected values of H50Q at 25°C for the diffuse phenotype was 44%, 42%, and 39% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of H50Q at 25°C for the plasma membrane phenotype was 20%, 26%, and 18% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of H50Q at 25°C for the aggregation phenotype was 36%, 32%, and 24% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of G51D at 25°C for the diffuse phenotype was 100%, 98%, and 99% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of G51D at 25°C for the plasma membrane phenotype was 0%, 0%, and 0% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of G51D at 25°C for the aggregation phenotype was 0%, 2%, and 1% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of A53E at 25°C for the diffuse phenotype was 55%, 48%, and 49% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of A53E at 25°C for the plasma membrane phenotype was 19%, 0%, and 31% for 12hrs, 18hrs, and 24hrs, respectively. The expected values of A53E at 25°C for the aggregation phenotype was 27%, 25%, and 19% for 12hrs, 18hrs, and 24hrs, respectively.

 

Figure 11. Toxicity of new familial mutants with ulp1ts and smt3ts

Spotting assay with five-fold serial dilutions of yeast with H50Q, G51D, and A53E in the ulp1ts strain (excess SUMO) or smt3ts strain (deficient SUMO) in on repressive (SC-Ura glucose) and inducive (SC-Ura galactose) media (n=6). There is effect of the strains at 30°C and no effect of the strains at 25°C.

 

Figure 12. Microscopy of new familial mutations in ulp1ts

The microscopy images shown are of the dominant phenotype observed for H50Q, G51D, and A53E at 25°C (normal SUMO levels) and 30°C (excess SUMO levels) in the ulp1ts strain. The images were taken at 12, 18, and 24 hours after induction in SC-Ura galactose (n=5).

 

Figure 14. Microscopy of new familial mutations in smt3ts

The microscopy images shown are of the dominant phenotype observed for H50Q, G51D, and A53E at 25°C (normal SUMO levels) and 30°C (deficient SUMO levels) in the smt3ts strain. The images were taken at 12, 18, and 24 hours after induction in SC-Ura galactose (n=5).

 

CHAPTER 3

EXAMINING SUMOYLATION AND PHOSPHORYLATION

 

Experimental set-up

My next goal was to look at the effects of SUMOylation and phosphorylation together on α-synuclein. These experiments were done in collaboration with Yoan Ganev ’19. Phosphorylation occurs at two main sites of α-synuclein: serine 87 or serine 129 (Fujiwara, 2002).  In this experiment, I either blocked or mimicked phosphorylation on α-synuclein mutants that already had SUMOylation blocked. To block phosphorylation, I mutated the serine (S) to an alanine (A), which cannot be phosphorylated (S129A and S87A/S129A). To mimic phosphorylation, I mutated serine (S) to an aspartic acid (D) due to its chemical similarity to phosphorylated serine (S129D and S87D/S129D). Next, I used the serial dilution spotting, fluorescence microscopy, and Western blot assays to test the effects of blocked SUMOylation in addition to either mimicked or blocked phosphorylation. 

 

Blocked phosphorylation counteracts the toxicity of blocked SUMOylation

First, I looked at the change in toxicity when I altered the levels of phosphorylation (Figure 16a). On the galactose plate, I compared the growth of the SUMOylation blocking mutation (K96R/K102R) to WT. Previously in chapter 1, K96R/K102R exhibited less growth compared to WT (Figure 8a). For this chapter, I used K96R/K102R as the control to compare the effects of altered phosphorylation levels and blocked SUMOylation. When I blocked phosphorylation and SUMOylation together on the single site (S129A/K96R/K102R), I observed better yeast growth compared to when I blocked SUMOylation alone (K96R/K102R) on α-synuclein (lane 4). Further, phosphorylation blocked at both sites of α-synuclein (S87A/K96R/K102R/S129A) exhibited better growth compared to K96R/K102R (lane 5), but the growth was not as pronounced as when I blocked phosphorylation on the single site. When I mimicked phosphorylation and blocked SUMOylation (S129D/K96R/K102R and S87A/K96R/K102R/S129A) on α-synuclein (lane 6 and 7), there was similar levels of yeast growth compared to blocked SUMOylation (K96R/K102R) alone. The glucose plates showed equal growth in each lane to account for equal amounts of yeast and even cell dilutions. 

 

Phosphorylation blocks reduces foci and phosphorylation mimics increase diffusion

Next, I qualitatively compared the localization patterns between the phosphorylation and SUMOylation mutants of α-synuclein in yeast. First, I compared localization of blocking SUMOylation (K96R/K102R) to WT. As shown in chapter 1, blocked SUMOylation leads to increased aggregation (Figure 9a). Next, I compared the SUMOylation and phosphorylation blocked mutations (K96R/K102R/S129A and S87A/K96R/K102R/S129A) to blocked SUMOylation alone. In figure 17a, Blocked phosphorylation and SUMOylation leads to a decrease in cells with aggregation after 18 hours. Finally, I compared the blocked SUMOylation and mimicked phosphorylation mutations (K96R/K102R/S129D and S87D/K96R/K102R/S129D) to blocked SUMOylation alone. Here, mimicked phosphorylation and blocked SUMOylation on α-synuclein increased diffusion after 18 hours (Figure 17a). 

I next scored the α-synuclein localization for the 5 microscopy trials. In Appendix A (p. 97-98), I graph the percentage of cells that display the three major localization patterns (diffuse, halo, foci/aggregation) for each mutant across the time points. In this case, the results for the single and double mutations where I blocked and mimicked phosphorylation were combined (Figure 17b). I then performed a chi-square test for goodness of fit to evaluate if the α-synuclein localization of the mutants were significantly different from blocking SUMOylation alone (K96R/K102R). The expected values of K96R/K102R for the diffuse phenotype was 43%, 17%, 22%, and 22% for 6hrs, 12hrs, 18hrs, and 24hrs, respectively.  The expected values of K96R/K102R for the plasma membrane phenotype was 45%, 46%, 60%, and 71% for 6hrs, 12hrs, 18hrs, and 24hrs, respectively. The expected values of K96R/K102R for the aggregation phenotype was 12%, 37%, 18%, and 7% for 6hrs, 12hrs, 18hrs, and 24hrs, respectively. Mimicked phosphorylation with blocked SUMOylation of α-synuclein led to a significant decrease in aggregation at 12 and 18 hours (p=<.05) and, instead, had a significant increase in diffusion at 6, 12, and 18 hours (p=<.01). When I blocked phosphorylation, there was a significant decrease in aggregation at 12 and 18 hours (p=<.05), and I observed a trend for higher percentages of membrane bound and diffuse cells.  

 

Amount of α-synuclein expressed is inconclusive

In figure 16b, I show a Western blot of WT, the mutation where we blocked SUMOylation, and the mutations where we either blocked or mimicked phosphorylation with blocked SUMOylation. The anti-α-synuclein antibody was used to tag α-synuclein protein. Anti-PGK antibody was used as a control to make sure there was equal amounts of yeast protein loaded. After three trials, each Western blot was inconsistent in terms of the amount of α-synuclein expression and the thickness of the control bands. 

 

a. Toxicity

 

b. Protein Expression

 

 

 

 

Figure 16. Toxicity and protein expression of phosphorylation mutations

(a) Spotting assay with five-fold serial dilutions of yeast with Vector, WT-eGFP, K96R/K102R, K96R/K102R/S129A, S87A/K96R/K102R/S129A, K96R/K102R/S129D, and S87D/K96R/K102R/S129D, and GFP in repressive (SC-Ura glucose) and inducive (SC-Ura galactose) media (n=6). 

(b) Western blot at 24 hours after α-synuclein expression for WT, K96R/K102R, S87A/K96R/K102R/S129A, and S87D/K96R/K102R/S129D. The anti-α-synuclein and anti-PGK loading control was used (n=3).

 

a. Live Cell Imaging

 

b. Quantification

 

Figure 17. Microscopy and quantification of phosphorylation mutations

(a) The live cell images shown are of the dominant phenotype observed for WT, K96R/K102R, K96R/K102R/S129A, S87A/K96R/K102R/S129A, K96R/K102R/S129D, and S87D/K96R/K102R/S129D variants over 24hrs. The images were taken at 6, 12, 18, and 24 hours after induction in SC-Ura galactose (n=5). 

(b)The time course quantification for the dominant phenotypes shown by WT, K96R/K102R, S87A/K96R/K102R/S129A, and S87D/K96R/K102R/S129D variants in a bar graph (n=4). In this graph the data for the single and double mutants for phosphorylation were combined.

 

CHAPTER 4

EXAMINING SUMOYLATION AND THE FAMILIAL MUTANTS

 

Experimental Set-up

My next goal was to evaluate the how the modifications effect the familial mutations. Mutation creation was done in collaboration with Joe Mountain ’20 and Ariane Balaram ’20. The experiments were done in collaboration with Yoan Ganev ’19. The familial mutations of α-synuclein (A30P, E46K, H50Q, G51D, A53T, and A53E) all lead to early onset Parkinson’s disease. In this experiment, I used the mutations in chapter 3, which blocked SUMOylation and altered the levels of phosphorylation, but now combined them with the familial mutations. In one case, I combined each familial mutation with the SUMOylation blocking mutation (K96R/K102R) and the phosphorylation blocking mutation (S87A/S129A). In the other case, I combined each familial mutation with the SUMOylation blocking mutation (K96R/K102R) and the phosphorylation mimicking mutation (S87D/S129D). To test the effects of combining the familial mutations with altered levels of SUMOylation and phosphorylation in budding yeast, I used the serial dilution spotting, fluorescence microscopy, and Western blot assays. 

 

Blocked SUMOylation Increases A53T Toxicity

First, I looked at the change in toxicity when I blocked SUMOylation in combination with the familial mutations of α-synuclein (Figure 18). As shown in chapter 1, blocked SUMOylation on WT α-synuclein exhibited more toxicity compared to WT (Figure 8a). In figure 18, I compared each familial mutation to the same familial mutation with blocked SUMOylation.  H50Q, G51D, A53E, A30P, and E46K all had similar levels of yeast growth when SUMOylation was or was not blocked. In contrast, blocked SUMOylation in combination with A53T exhibited less cell growth compared to A53T with normal levels of SUMOylation. 

Blocked SUMOylation and Phosphorylation Leads to Better Growth for E46K

Next, I observed the change in toxicity when I blocked both SUMOylation and phosphorylation with each familial mutation of α-synuclein. In chapter 3, I observed better growth when both phosphorylation and SUMOylation were blocked compared to when SUMOylation was blocked alone (figure 16a). In figure 19, I compare each SUMOylation-deficient familial mutation to the same familial mutation with SUMOylation and phosphorylation blocked. When I blocked SUMOylation and phosphorylation on E46K, it grew more than when I blocked SUMOylation alone with E46K. In the case of A53T and H50Q, there might be slightly better growth when SUMOylation and phosphorylation is blocked.  All other familial mutations with SUMOylation and phosphorylation blocked had similar growth to the same SUMOylation-deficient familial mutation.

 

Blocked SUMOylation and Mimicked Phosphorylation Leads to Less Growth for A53T

In the next goal, I observed the change in toxicity when I blocked SUMOylation and mimicked phosphorylation with each familial mutation. In chapter 3, blocked SUMOylation and mimicked phosphorylation on WT α-synuclein led to similar levels of growth compared to blocked SUMOylation alone (Figure 16a). In figure 20, I compare each SUMOylation-deficient familial mutation to the same familial mutation but with SUMOylation blocked and phosphorylation mimicked. When I blocked SUMOylation and mimicked phosphorylation on A53T, it grew less than when I blocked SUMOylation alone on A53T. In the case of G51D, there was slightly less growth with blocked SUMOylation and mimicked phosphorylation. All other familial mutations had similar growth to the same familial mutations with blocked SUMOylation and mimicked phosphorylation.

 

Familial Mutations Dominate over Localization Pattern when Blocking SUMOylation

Next, I qualitatively compared the localization patterns between the familial mutations with normal levels of SUMOylation and the SUMOylation-deficient familial mutations (Figure 21). As shown in chapter 1, blocked SUMOylation on WT α-synuclein exhibited aggregates even after 24 hours. SUMOylation-deficient familial mutants led to similar localization patterns as the familial mutants with normal levels of SUMOylation.  For example, A30P and G51D usually exhibit a diffuse pattern throughout the time course. When I blocked SUMOylation with A30P and G51D, I still saw cytoplasmic diffusion throughout the time course. E46K, H50Q, A53T, and A53E usually display some aggregates but eventually the α-synuclein reaches the membrane. Similarly, I observed some aggregates but eventual membrane binding when I blocked SUMOylation on these familial mutants. 

 

Blocked Phosphorylation on the Familial Mutations Leads to Diffusion

Next, I qualitatively compared the localization patterns between the familial mutations with blocked SUMOylation and phosphorylation to the familial mutations with blocked SUMOylation and mimicked phosphorylation. As shown in chapter 3, mimicked phosphorylation and blocked SUMOylation on WT α-synuclein exhibited cytoplasmic diffusion. Similarly, blocked SUMOylation and mimicked phosphorylation showed increased cytoplasmic diffusion in all the familial mutations (Figure 22).

In chapter 3, blocked SUMOylation and phosphorylation together on WT α-synuclein exhibited more membrane binding. Next, I blocked both modifications with the familial mutations. In A30P, the diffusion was preserved throughout the time course. G51D appeared to have diffusion mixed with some membrane binding at 24 hours. E46K, H50Q, A53T, and A53E all exhibited membrane binding at 24 hours. 

 

Figure 18. Toxicity of blocking SUMOylation with the familial mutants

Spotting assay with five-fold serial dilutions of yeast with the familial mutations (A30P, E46K, A53E, H50Q, G51D) when SUMOylation is blocked (K96R/K102R) or present on repressive (SC-Ura glucose) and inducive (SC-Ura galactose) media (n=6).

 

Figure 19. Toxicity of familial mutants with mimicked phosphorylation

Spotting assay with five-fold serial dilutions of yeast with the familial mutations (A30P, E46K, A53E, H50Q, G51D) when SUMOylation is blocked (K96R/K102R) and phosphorylation is at normal levels or deficient (S87A/S129A) on repressive (SC-Ura glucose) and inducive (SC-Ura galactose) media (n=6). 

 

Figure 20. Toxicity of familial mutants with blocked phosphorylation

Spotting assay with five-fold serial dilutions of yeast with the familial mutations (A30P, E46K, A53E, H50Q, G51D) when SUMOylation is blocked (K96R/K102R) and phosphorylation is at normal levels or deficient (S87A/S129A) on repressive (SC-Ura glucose) and inducive (SC-Ura galactose) media (n=6). 

 

Figure 21. Microscopy of familial mutants with mimicked phosphorylation

The microscopy images shown are of the dominant phenotype observed for the familial mutations (A30P, E46K, A53T, H50Q, G51D, A53E) with and without SUMOylation (K96R/K102R). The images were taken at 6, 12, 18, and 24 hours after induction in SC-Ura galactose (n=6).

 

Figure 22. Microscopy of familial mutants with blocked phosphorylation

The microscopy images shown are of the dominant phenotype observed for the familial mutations (A30P, E46K, A53T, H50Q, G51D, A53E) when SUMOylation is blocked (K96R/K102R) and phosphorylation is either mimicked (S87D/S129D) or blocked (S87A/S129A). The images were taken at 6, 12, 18, and 24 hours after induction in SC-Ura galactose (n=6).

 

CHAPTER 5

IS SUMOYLATION ALWAYS PROTECTIVE?

 

Experimental Set-up

My next goal was to understand the effects of blocked SUMOylation and altered levels of phosphorylation in the fission yeast model. Although fission yeast divides differently from budding yeast, they both share genes with higher eukaryotes and can be used to evaluate α-synuclein’s aggregation and toxicity. The Dr. DebBurman lab was the first lab to use fission yeast as a model organism to study Parkinson’s disease (Brandis, 2006). Unlike budding yeast, WT α-synuclein forms aggregates and is nontoxic in fission yeast (Brandis, 2006).  With the help of Alex Roman ’16 and Yoan Ganev ’19, I made SUMOylation blocking mutations (K96R, K102R, K96R/K102R) and mutations with altered phosphorylation (S129A, S87A/S129A, S129D, S87D/S129D) in fission yeast. I then used the serial dilution spotting and microscopy assays. 

 

Blocked SUMOylation leads to better growth than WT

First, I observed the effects of blocked SUMOylation on the growth of fission yeast. The fission yeast vector, pNMT1, contains a thiamine-repressible promotor. In this case, EMM-T media induces α-synuclein protein expression, while EMM+T media represses α-synuclein protein expression. Compared to the results in budding yeast, I see a different trend in fission yeast. On EMM-T media, I found that K96R, K102R, and K96R/K102R grew slightly more than WT α-synuclein (Figure 23a). There was uniform growth on repressive EMM+T media to show that the main effects are coming from the α-synuclein and its variants. 

 

Blocked SUMOylation leads to smaller aggregation

Next, I observed the live cell imaging of blocked SUMOylation in fission yeast over 48 hours of . In Figure 23b, WT α-synuclein showed large aggregates at 24 hours. The size of the aggregates decreased at 36 and 48 hours. In contrast, both K96R and K96R/K102R showed smaller aggregations at 24 hours compared to WT. At 36 and 48 hours, the mutants exhibited similar sizes of aggregates to WT α-synuclein. The K102R still displayed large aggregations but were smaller than WT at 24 hours. 

 

No change in toxicity when phosphorylation is altered

Next, I observed the change in toxicity when I altered the levels of phosphorylation in fission yeast (Figure 24). On the EMM-T, I compared the growth of the SUMOylation blocking mutation (K96R/K102R) to WT. As shown in figure 23a, I observed more growth of the blocked SUMOylation mutation (K96R/K102R) to WT. I observed no significant change in cell growth when I altered the levels of phosphorylation, either mimicked or blocked, compared to when I blocked SUMOylation alone. The EMM+T plate showed equal growth in each lane to account for even cell dilutions. 

 

a. Toxicity

 

b. Live Cell Imaging

 

 

Figure 23. Toxicity and microscopy of FY SUMOylation blocking mutations 

(a) Spotting assay with five-fold serial dilutions of yeast with Vector, GFP, WT-GFP, K96R, K102R, and K96R/K102R on repressive (EMM+T) and inducive (EMM-T) media (n=6). 

(b) The live cell images shown are of the dominant phenotype observed for WT, K96R, K102R, and K96R/K102R over 48hrs. The images were taken at 12, 24, 36, and 48 hours after induction in EMM-T (n=6).

 

Figure 24. Toxicity of phosphorylation mutations in fission yeast 

Spotting assay with five-fold serial dilutions of yeast with Vector, WT, K96R/K102R, K96R/K102R/S129A, S87A/K96R/K102R/S129A, K96R/K102R/S129D, and S87D/K96R/K102R/S129D, and GFP in repressive (EMM+T) and inducive (EMM-T) media (n=6). 

 

Discussion

In patients with Parkinson’s disease, different modifications can either contribute to or protect against pathogenesis of a disease. Two modifications, SUMOylation and phosphorylation, bind to the accumulated α-synuclein within the Lewy bodies of PD patients.  It is unclear whether these modifications contribute to α-synuclein dysfunction or stabilization. Thus, I sought out to unravel the purpose of α-synuclein SUMOylation and phosphorylation in yeast. I made mutations that alter the levels of SUMOylation and phosphorylation and then observed the localization, toxicity, and expression of α-synuclein. Using yeast, I found: 1) SUMOylation is protective 2) Phosphorylation is toxic in budding yeast 3) Familial mutations dominate over the effects of the modifications 4) SUMOylation is toxic in fission yeast. 

 

SUMOylation is protective

SUMOylation is thought to be protective and increase protein solubility, but these studies were only shown in-vitro and in a few in vivo studies. Based off these few studies, I hypothesized that SUMOylation is also protective in the living model organism of budding yeast. My data supported this hypothesis. Blocked SUMOylation on α-synuclein led to increased aggregation and toxicity compared to WT α-synuclein within budding yeast. 

This increased aggregation and toxicity when SUMOylation is blocked suggests that retention of normal levels of SUMOylation could allow for increased α-synuclein solubility and increased plasma membrane localization within yeast. Recently, more results confirm that SUMOylation inhibits the aggregation of α-synuclein and other proteins (Krumova et al. 2011; Abywardana & Pratt, 2015; Shahpasandzadeh et al., 2014). One study used protein semisynthesis to create SUMOylated α-synuclein, and it exhibited aggregation inhibition (Abywardana & Pratt, 2015). In this study, SUMOylation at K102 showed better aggregation inhibition compared to K96. In contrast, my research showed that K96 is a more heavily SUMOylated site than K102 due to greater toxicity with K96R and little to no toxicity with K102R. This decrease in toxicity with the K102R mutant might be due to inefficient blocking of SUMOylation. One study showed that K102R mutant in HEK293 cells were still slightly modified by SUMO and not completely SUMO-blocked (Dorval & Fraser, 2006).  

Other research has shown that the SUMO protein is a player in the autophagy-lysosome system that is activated to combat aggregated and accumulated protein (Wong et al. 2013; Vijayakumaran, Wong, Antony, & Pountney, 2015). In addition, SUMOylation helps sort α-synuclein into extracellular vesicles (Kunadt et al., 2015). These findings support SUMO’s protective role when associated with α-synuclein. 

When using the temperature sensitive strains (ulp1ts and smt3ts) with excess and deficient levels of SUMOylation, deficient levels of SUMOylation led to toxicity compared to excess levels of SUMOylation. Even G51D, which usually exhibits more cell growth than WT α-synuclein, exhibited toxicity. This data is consistent with another recent study which shows increased toxicity with deficient levels of SUMOylation using the same smt3ts strain (Shahpasandzadeh et al., 2014). Also, in this study, decreased SUMO levels increased aggregation formation in yeast. In contrast, I did not observe differences in the localization of the familial mutants of α-synuclein with excess or deficient levels of SUMO. In this case, the familial mutant localization patterns dominated over the changes in SUMOylation levels. In addition, when using the smt3ts strain, I noticed an abundance of dead cells without fluorescence. These dead cells might have been the cells affected by deficient SUMO. It is possible that I only quantified cells that were outliers and had not been affected by the smt3ts strain. This would explain why there was no significant difference in the fluorescence for the smt3ts strain when clear differences in toxicity were found. The sudden loss of SUMOylation might be so toxic to the cells that they died. These cells might have also acted on different mechanisms of defense to survive. One possible phenomenon is called loss of plasmid in which the yeast ejects the plasmid as a strategy to decrease toxicity.  Ejection of the plasmid would mean decreased GFP fluorescence and limitations on the quantifications I performed.  

 

Phosphorylation is toxic in budding yeast

In PD patients, there is a significant increase in phosphorylation of α-synuclein. 3 years ago, when I started this project, the role of phosphorylation on α-synuclein still had controversies. Based on fly (Chen & Feany, 2005) and human neuroblastoma studies (Smith et al., 2005), I hypothesized that phosphorylation was toxic and leads to more aggregation and that SUMOylation might counteract this toxicity. I found evidence for and against this hypothesis.  

First, I found blocked phosphorylation and SUMOylation leads to more protection, while mimicked phosphorylation with blocked SUMOylation leads to similar levels of toxicity compared to SUMOylation-deficient α-synuclein. Since there is more growth when phosphorylation was not present, these findings support my hypothesis that phosphorylation is toxic, but this protection also occurs while SUMOylation is blocked. This demonstrates how blocked phosphorylation counteracts the toxicity seen when SUMOylation is blocked. In contrast, one study done with temperature sensitive defects in SUMO with the blocking or mimicking of phosphorylation found that mimicking phosphorylation counteracts the toxicity of deficient SUMOylation (Shahpasandzadeh et al., 2014). In my research, mimicked phosphorylation leads to no difference in the toxicity. One finding has shown that their results on degradation pathways used for α-synuclein clearance are also different from the Shahpasandzadeh study (Arawaka, Sato, Sasaki, Koyama, & Kato, 2017). One possible explanation for this contrast to my findings and the findings in the Arawaka study is due to the Shahpasandzadeh study’s use of the overexpression of human kinases, GRK5 and PLK2, which phosphorylates S129. This could possibly change the phosphorylation levels and degradation pathways used in yeast and, therefore, create differences in regulation of mimicked phosphorylation.  

Further in my study, blocked phosphorylation and SUMOylation leads to decreased aggregation, but no significant increase in diffusion or membrane binding. This again demonstrates how blocked phosphorylation might counteract the effects of increased aggregation when SUMOylation is blocked. Interestingly, mimicked phosphorylation also decreases aggregation within the cell when SUMOylation was blocked, but, in this case, there was also a significant increase in diffusion. These findings are consistent with recent in vitro studies that demonstrate how α-synuclein with phosphorylation mimics at S87 unbinds from vesicles (Kumar, Schilderink, Subramaniam, & Huber, 2017). Another paper published from our lab also found that the S129D phosphorylation mimic alone decreased plasma membrane association in budding yeast (Fiske et al., 2011). In the past, there has been a recurring connection between diffusion and protection. Specifically, there are multiple occasions where diffusion within the budding yeast is correlated with protection from toxicity in the yeast (Fares et al., 2014; Tembo, Thesis, 2015; Ong, Thesis, 2017; Jones, Thesis, 2018). There still needs to be more research on how or why cytoplasmic diffusion induces protection in yeast. In my findings, mimicked phosphorylation did not lead to an increase in protection, but it also did not lead to further toxicity. Although there was no significant protection, the diffusion itself suggests that the mimicking of phosphorylation could possibly be connected to a type of protective mechanism in yeast against α-synuclein toxicity, but this was not demonstrated in this research. 

To summarize these findings, blocking of phosphorylation leads to more protective properties and less aggregation, while mimicking of phosphorylation also leads to diffusion and no significant change in cell growth.  More recent findings have shed light onto why my results, in addition to the results of other studies, are controversial to each other. One characteristic of PD is mitochondrial impairment. The mitochondria functions in calcium homeostasis, and, therefore, impairment leads to increased influx of extracellular calcium. GRK5, a kinase that phosphorylates α-synuclein, is activated by calcium and calmodulin to increase phosphorylation at S129 of α-synuclein in PD. After, the phosphorylated α-synuclein is moved to the proteasome pathway for degradation, while the lysosomal pathway is inhibited to promote more ubiquitin-proteasomal targeting of α-synuclein. Later, the proteasomal targeting becomes ineffective, but there is still phosphorylation to target α-synuclein for proteasomal degradation. As a result, the α-synuclein within PD patients are highly phosphorylated, and there is an accumulation of phosphorylation (Arawaka, Sato, Sasaki, Koyama, & Kato, 2017). 

This model demonstrates that phosphorylation might have a protective function, but the accumulation of phosphorylation possibly causes future problems for the normal functioning of the proteasomal degradation pathway. These findings might give reason to why there are contradictory findings on whether phosphorylation is protective or toxic in different model organisms. It could be both toxic and protective depending on the functioning of the proteasomal pathway. 

 

Familial mutants dominate over effects of the modifications

Familial mutations of α-synuclein give rise to early onset PD. My goal was to understand whether the modifications of SUMOylation and phosphorylation could alter the effects of the familial mutations. When SUMOylation is blocked with the familial mutations, only A53T becomes more toxic; All other familial mutations showed no change in toxicity. Recent research shows that there is a sevenfold increase in SUMOylation of α-synuclein with the old familial mutations in vitro (Rott et al., 2017). This gives evidence that the mutated α-synuclein is getting SUMOylated to combat the insoluble α-synuclein, but to no help as my research shows there is no change in cell growth for most mutations. 

Instead, the in vitro study demonstrates that this excessive SUMOylation leads to increased aggregation (Rott et al., 2017). In contrast, using budding yeast, I did not observe a significant increase or decrease in aggregation when SUMOylation is blocked with the familial mutations. Instead, the normal phenotypes of each familial mutation dominated over any change in SUMOylation. For example, A30P and G51D usually exhibit cytoplasmic diffusion within the cell, and they retain this diffusion when SUMOylation is blocked. This difference in aggregation formation might be due to the study’s use of PIAS2, an enzyme that SUMOylates protein, while I used budding yeast with natural SUMOylation ligases. 

Blocked SUMOylation and phosphorylation leads to decreased toxicity of the E46K mutant compared to when only SUMOylation is blocked. The A53T and H50Q mutants also exhibited slightly more cell growth. Recent research has come out showing that E46K α-synuclein is the most S129 phosphorylated mutant of α-synuclein (Íñigo-Marco et al., 2017; Mbefo et al., 2015). My research gives further evidence that there is a connection between E46K and phosphorylation in the progression of PD. This finding also supports my previous finding that blocked phosphorylation counteracts the toxicity of blocked sumoylation.

Blocked SUMOylation and mimicked phosphorylation leads to increased toxicity of the A53T mutant compared to when only SUMOylation is blocked. The G51D mutant might also have slightly less growth. A53T α-synuclein is also highly phosphorylated, similar to E46K (Mbefo et al., 2015). It is still unknown why these particular mutations are sensitive to phosphorylation mimics with SUMOylation blocks. It is interesting that G51D has less growth because it usually displays increased yeast growth even through it is one of the familial mutations that leads to early onset PD (Tembo, Thesis, 2014). Phosphorylation and SUMOylation might be a key to understanding why G51D is protective in yeast. Overall, the toxicity findings show that phosphorylation might have differential effects depending on the familial mutation and their propensity to be phosphorylated. 

When blocking phosphorylation, I observed no significant change in α-synuclein localization, but mimicking phosphorylation led to an increase in cytoplasmic diffusion. One study looked at the aggregation of the A53T mutant and compared it to A53T with S129A double mutant α-synuclein and found no difference in aggregation similar to my results (Arawaka, Sato, Sasaki, Koyama, & Kato, 2017). Interestingly, another study found that there is a loss of phosphorylation at S129 following the binding of WT, A30P, and A53T α-synuclein to the membrane of neuron terminals (Visanji et al., 2011). This would explain why there is less membrane binding and more cytoplasmic diffusion when phosphorylation is mimicked. There is no loss of phosphorylation to allow for proper membrane binding. 

 

SUMOylation is toxic in fission yeast

Fission yeast has given insights into cell cycle functioning, control of telomere length, DNA repair, and more (Fantes & Beggs, 2000; Tanaka et al., 1999; Davis & Smith, 2001; Brandis et al., 2006). Similar to budding yeast, fission yeast folds and controls protein like humans. Unlike budding yeast, WT α-synuclein forms aggregates in fission yeast forms, while WT α-synuclein in budding yeast eventually binds to the membrane. 

Blocked SUMOylation in fission yeast leads to less toxicity and a decrease in aggregation size. These findings go against my hypothesis that SUMOylation is protective. Brandis et al. (2006) pointed out that the ability of plasma membrane localization, which is diminished in fission yeast, might be key for toxicity. This would explain why there is no significant toxicity when altering phosphorylation and SUMOylation levels in fission yeast.

The SUMO protein could hold a different function in fission yeast from budding yeast. The SUMO protein itself can have multiple different functions depending on the context. For example, the SUMO protein can modify cellular processes, function in cellular stress responses, and allow for protein solubility (Hay, 2005). Therefore, these differences in function could give rise to different effects of SUMOylation in fission yeast versus budding yeast when blocked on α-synuclein. One example of how a modification can have different effects similar to SUMOylation is phosphorylation’s effects in rats versus Drosophila. Blocked phosphorylation in rats is toxic, while blocked phosphorylation in Drosophila is protective (Chen & Feany, 2005; Gorbatyuk et al., 2008).   

It is possible that SUMOylation is not always protective. In Huntington’s disease, SUMO-modified proteins accumulate in the striatum of patients (O’Rourke et al., 2013). Reduction of SUMO ligase, PIAS1, using miRNA stops Huntingtin protein accumulation in mice (Ochaba et al., 2016). Overexpression of PIAS1 in mice leads to increased Huntingtin protein accumulation. These findings suggest that SUMOylation might promote the pathology of Huntington’s disease. Similarly, it is possible that SUMOylation is also promoting PD-like pathology in fission yeast. Given these complex possibilities of the role of SUMOylation, it should not surprise us that there are different effects in budding yeast versus fission yeast. Fission yeast and budding yeast are 300 to 600 million years apart in evolution. More research must be performed to understand the role of α-synuclein SUMOylation in fission yeast. 

 

Aggregation does not cause toxicity

It has been hypothesized that aggregation of α-synuclein leads to toxicity. My research does not always coincide with this hypothesis. For example, when I blocked SUMOylation at K102, there was an increase of aggregation, but no significant toxicity. In addition, with deficient levels of SUMO protein with the G51D familial mutation, there was increased toxicity, but no increase in aggregation. Instead, there was cytoplasmic diffusion throughout the cells. 

One possible reason of these differences in toxicity with or without aggregates is due to the presence of soluble oligomers. The A30P familial mutation forms soluble oligomers, but leads to early onset PD (Sharon et al., 2003). For prion diseases, Alzheimer’s disease, and Huntington’s disease, there is evidence that the aggregates do not cause toxicity themselves (Sisodia et al., 1998; Jucker & Walker, 2013; Taylor, Hardy, & Fischbeck, 2002). These examples demonstrate that the aggregates might not cause toxicity themselves, but toxicity might stem from soluble oligomers. 

 

Limitations of my study

Although yeast are powerful model organisms in the study of Parkinson’s disease, they contain limitations because they do not fully model the dopaminergic neurons in PD patients. This limitation is especially apparent as they do not naturally contain α-synuclein or dopamine. In addition, yeast assays can be interpreted in multiple different ways. For example, in the live-cell imaging assay, some of the aggregates within yeast could possibly be accumulated α-synuclein bound to vesicles (Soper et al., 2008). Also, toxicity of α-synuclein with the serial-dilution spotting assay should be quantitatively measured versus qualitatively, as there is always the possibility that the cells are not dying but getting smaller. 

 

Future studies

My thesis raises several questions: 1) How is SUMOylation increasing solubility of α-synuclein molecularly? 2) Why is there an increase of cell death with deficient levels of SUMO using the smt3ts strain? 3) How does blocked phosphorylation counteract the toxicity and aggregation of blocked SUMOylation? 4) What are the mechanisms of dysfunction for each of the familial mutants in PD? 5) Is SUMOylation always acting as a protective modification?

In order to look at the molecular mechanisms underlying SUMOylation and phosphorylation, I would use Nuclear magnetic resonance microscopy to determine α-synuclein oligomer levels (Ghosh et al., 2014). I would also use CD spectroscopy to view how α-synuclein structure has changed when SUMOylated or phosphorylated (Stöckl, Zijlstra & Subramaniam, 2013). In the future, I would also like to improve my Western blot skills to observe the amount of α-synuclein made with the SUMOylation and phosphorylation modifications. In this case, I could also understand how α-synuclein modifications affect different aspects of the cell by using antibodies against ER-Golgi trafficking markers and nitrosative stress (Cooper et al., 2006; Tardiff et al., 2013). I was not successful in performing a Western blot on fission yeast. The changes in α-synuclein accumulation in fission yeast could give insight into the role of SUMOylation in this model organism. In addition, it would be interesting to understand how sumoylation and phosphorylation effect other modifications such as nitration, acetylation, and glycation. 

 

Conclusion

My Findings

This thesis provided insight into the role of sumoylation and phosphorylation in Parkinson’s disease. Through this project, I successfully created and characterized mutants that altered levels of SUMOylation and phosphorylation. I gave more evidence for the protective role of SUMOylation and information on the potential toxic and protective roles of phosphorylation. 

 

Importance of α-Synuclein Modifications

In PD, α-synuclein protein is misfolding, coming out of solution, and forming aggregates within the brain. In both sporadic and familial PD, further research should be performed on how α-synuclein is misfolding and how it is connected with toxicity and the death of dopaminergic neurons. Some molecules have the potential to bind to α-synuclein and rescue the protein from aggregation. Other molecules could bind and lead to increased aggregation and later toxicity. My research and the research of other labs show how further studies into the complex pathways of α-synuclein modifications could give insight into possible treatments and therapies for the patients suffering with PD.

 

Parkinson’s Disease: Together We Can Win

As life expectancy increases, more people will be affected by neurodegenerative diseases like Parkinson’s disease. My family and I have personally been touched by this debilitating disease. Although many have died to PD, their lives give us insight on how to save other lives. Therefore, we should not lose hope in the face of PD. Every problem has its solution, and we can find that solution with the help of research done together. I know the PD patients, caregivers, and family members are cheering us on. They can’t wait for a cure.  

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Appendix A

 

 

 

 

 

 

Figure 25. Quantification of phosphorylation mutations

The time course quantification for the dominant phenotypes shown by WT, K96R/K102R, S129A, S87A/K96R/K102R/S129A, S129D, and S87D/K96R/K102R/S129D variants in a bar graph (n=4). 

 

Appendix B

 

 

 

 

 

 

Figure 26. protein expression of SUMOylation blocking mutations 

Western blot at 24 hours after α-synuclein expression for WT, K96R, K102R, and K96R/K102R. The anti-α-synuclein and anti-PGK loading control was used (n=3). 

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