Summary

Parkinson’s disease (PD) is a progressive neurodegen­erative disorder, characterized by aggregation of the α-synuclein protein into Lewy bodies and a loss of dopaminergic neurons, leading to a deficiency in the dopamine neurotransmitter. There are two different types of PD: idiopathic (environmental) and familial (genetic). The focus of this research is familial PD which is known to be associated with seven different genes. One of these genes is α-synuclein, which codes for the α-synuclein protein. The goal of this research is to create yeast models of α-synuclein with each model expressing either the K6R/K10R or K6Q/K10Q mutations on a single familial mutant of the protein. We hypothesize that a yeast model expressing the K6R and/or K10R mutations on a familial mu­tant version of α-synuclein will experience increased aggregation of the protein into Lewy-body-like aggregates (compared to non-mutat­ed α-synuclein). This is because the K6R and K10R mutations block acetylation and promote the glycation of α-synuclein, a post-trans­lational modification linked with increased α-synuclein aggregation. In a yeast model expressing the K6Q and/or K10Q mutations and a familial mutant, α-synuclein will be acetylated but not glycated, leading to decreased aggregation of the protein and a decrease in Lewy-body-like aggregates. We attempted, but failed, to create the K6R/K10R mutation on the familial mutants: E46K, H50Q, G51D, and A53E yeast models. We successfully mutated K6R on a A53E tem­plate and K6Q/K10Q on a A30P template. These yeast models can be utilized in future PD research to investigate the effect the mutations have on α-synuclein and PD.

Introduction

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that includes a variety of motor and non-motor symptoms. It was first introduced to the scientific world by James Parkinson in 1817; however, the disease is still incurable (Parkinson, 2002). Motor symp­toms include tremors at rest, postural instability, muscular rigidity, flexed posture, and freezing of gait (Khan et al., 2017). Non-motor symptoms include autonomic instability, cognitive dysfunction, neuropsychiatric dis­orders, and sensory and sleep abnormalities. The pathological hallmarks of this disease include theloss of dopaminergic neurons, which leads to a deficiency in the dopamine neurotransmitter, and the presence of Lewy bodies, which are aggregates of the protein α-synuclein (Schulz-Schaef­fer 2015). There are two major categorizations of PD: idiopathic and familial. Idiopathic PD is not heritable or genetic, and although the exact pathology of this form isn’t known, multiple etiological environmental factors have been identified, including heroin use, exposure to pesticides, consumption of well-water, and head trauma (Schiesling et al., 2008). Familial PD is genetic, with at least seven genes known to be associated with its pathology (α-synuclein, Parkin, PINK1, UCH-L1, DJ-1, LRRK2, and ATP13A2).

The α-synuclein protein plays a particularly important role in the development and progression of PD. α-synuclein’s function is to assist the neuron’s response to stress and to regulate the transmission of neuronal signals (Benskey, Perez, and Manfredsson, 2016). In PD, α-synuclein accumulates into Lewy bodies that can be found in some remaining neurons. These Lewy bodies form aggregates, which make it so that the protein can no longer function normally. This association between α-synuclein and PD has a genetic basis. There are six mutant forms of this protein: A53T, A30P, E46K, H50Q, G51D, and A53E. α-sy­nuclein’s protein sequence is separated into three different categories: the N-terminal amphipathic region, the central NAC domain (non-amyloid b-component) and the C-terminal, which is the acidic region that contains phosphorylation sites. All six of the familial mutations of α-synuclein are located in the N-terminal amphipathic region of the protein (Fujioka et al., 2014). The mutant form of α-synuclein that has an amino acid change on the 53rd codon of the 140 codon-long gene, from Alanine to Threonine (A53T), has been linked to PD because it causes increased protein ag­gregation (Polymeropoulos et al., 1997). Since this initial genetic variation in α-synuclein was first found and connected with PD in 1997, additional familial mutant forms of α-synuclein linked to PDhave emerged. These familial mutants have also been shown to increase protein aggregation in α-synuclein, leading to PD. These familial mutants of a-synuclein include: an amino acid change on the 30th codon of the α-synuclein gene from Alanine to Proline (A30P) (Krüger et al., 1998), a change from Glutamate to Lysine on the 46th codon (E46K) (Zarranz et al., 2004), a change from Histidine to Glutamine on the 50th codon (H50Q) (Appel-Cresswell et al., 2013), a change from Glycine to Aspartate on the 51st codon (G51D), (Lesage et al., 2013), and a change from Alanine to Glutamate on the 53rd codon (A53E) (Pasanen et al., 2014). All of these described muta­tions have been linked to familial PD pathophysiology. These mutations have been investigated in cells to examine how they impact α-synuclein’s aggregation and role in MS (Lázaro et al., 2014). It has been found that the variants discussed impact aggregation by encouraging α-synuclein oligomerization.

In addition to these familial mutants, two additional double mu­tations in α-synuclein have been hypothesized to be involved in PD: a Ly­sine to Argenine mutation on codons 6 and 10 (K6R/K10R), and a Lysine to Glutamine mutation on the same codons (K6Q/K10Q) (De Oliveira et al., 2017). The K6R/K10R mutations block the acetylation of α-synuclein, which in turn promotes α-synuclein aggregation into Lewy bodies and the death of dopaminergic neurons in the substantia nigra. These mutations also result in the promotion of the glycation of α-synuclein, which has been linked to an increase in α-synuclein toxicity, a hallmark of PD (Mur­onetz et al, 2017). On the other hand, the K6Q/K10Q mutations promote the acetylation of α-synuclein, which has been shown to be protective against PD. Although the discussed familial mutations (A30P, E46K, H50Q, G51D, A53T, and A53E) and the acetylation/glycation mutations K6R/K10R have been linked to PD pathology, and the K6Q/K10Q muta­tions have been linked to the protection against α-synuclein aggregation, the interactions of these acetylation/glycation mutations and the familial mutations are unknown. We hypothesize that α-synuclein will experience increased aggregation into Lewy-body-like aggregates in a yeast model of PD expressing the K6R and/or K10R mutations on any of the familial mu­tants of a-synuclein. This is because the K6R and K10R mutations block acetylation and promote the glycation of α-synuclein, both of which have been linked to increased toxic accumulation of the misfolded protein. We also hypothesize that α-synuclein aggregation will decrease in a yeast model expressing the K6Q and/or K10Q mutations and any of the familial mutants, leading to a decrease in Lewy-body-like aggregates.

Yeast (Saccharomyces cerevisiae) is a useful model organism for studying many neurodegenerative diseases, including PD. Yeast allow for relatively easy knockout and isolation of specific genes and can be used to observe and experiment with many of the same processes (including protein activity, packaging, manufacturing, and degradation) that happen in human cells. These observations can be readily applied to the processes underlying human diseases (Kain, 2008). Yeast also multiply quickly and the creation of a transgenic cell line is relatively easy. Previous work (including that from our laboratory) has shown that yeast (S. cerevisiae) are an effective and useful model of α-synuclein aggregation and toxicity in PD (Fiske et al., 2011). For example, one could examine the impact of these mutations on the growth of yeast cells using a spotting assay (Duennwald, 2012). One could also perform immunostaining with an α-synuclein antibody and examine the distribution and accumulation of α-synuclein in these mutant models (Zabrocki et al, 2005). Additionally, one could also examine how mutant α-synuclein interacts with other proteins in yeast. Expressing α-synuclein in yeast allows scientists to study its effects; as a result of this study, it has been found that the misfolding of the α-synuclein protein can have important neurodegenerative outcomes (Outeiro and Lindquist, 2003).

In this study, we set out to create yeast models of α-synucle­in, each expressing the K6R/K10R or K6Q/K10Q mutations of a single familial mutant. To create these models, we first ran a mutagenesis polymerase chain reaction (PCR) to mutate familial α-synuclein mutant plasmid models with K6R/K10R or K6Q/K10Q using mutagenesis prim­ers. Upon confirming a successful mutagenesis reaction using gel electro­phoresis, we then transformed the α-synuclein plasmids into E. coli cells to replicate the plasmids. The plasmids were then purified and sequenced and transformed into yeast. These yeast models will allow researchers to investigate the interactions of the acetylation/glycation mutations and the familial mutations.

Results

Project Layout and Design

In this project, we mutagenized familial mutants of α-synucle­in. Figure 1A shows the layout of the α-synuclein gene that was used (including the locations of the mutations that we worked with), as well as the attached green fluorescent protein (GFP) gene. The α-synuclein gene and GFP were on the P-YES2 plasmid. To mutate α-synuclein, we first started with a familial mutant template (one of the six familial mutations of α-synuclein discussed), and ran a mutagenesis polymerase chain reaction (PCR). This PCR reaction used forward (FP) and reverse (RP) primers with the desired mutation, as shown in Figure 1B. To confirm that the primers used would work properly, forward and reverse primers were run on a primer check gel using gel electrophoresis (GEP) (Figure 1D). The mutagenized α-synuclein P-YES2 plasmids were then transformed into E. coli to replicate them and then purified and sequenced. The se­quenced plasmids were then transformed into yeast. The overall layout of the progression of our experiments is shown in Figure 1C.

Mutagenesis PCR and GEP

To mutate the α-synuclein plasmids, a mutagenesis PCR reac­tion was run with the primers shown in Figure 1B. The first PCR reaction used the H50Q, G51D, and A53Ε α-synuclein familial mutant templates and the K6R/K10R primers. The PCR products were then run on a gel using GEP to look for the presence of PCR product (Figure 2). If the PCR reaction ran successfully, thicker bands in lanes 5, 7, and 9 would be observed (when compared to lanes 4, 6, and 8). The GEP layout in Figure 2A is an idealized version of the gel that depicts what the GEP would look like if successful. The actual GEP is shown in Figure 2B. This reaction was attempted twice, and the GEP of the second PCR is shown in Figure 3, with Figure 3A being the idealized gel (having thicker bands in lanes 4, 7, and 10) and Figure 3B showing the actual GEP.

An additional PCR reaction was run using the H50Q and E46K templates with the K6R/K10R primers. The GEP for this PCR is shown in Figure 4, where Figure 4A depicts the idealized version of the gel (note the distinct bands in lanes 8 and 10), and Figure 4B shows the actual GEP.

Another mutagenesis PCR was attempted (by A. Dailey and V. Miller) using the H50Q, G51D, and A53E templates and the K6R primers. The GEP for this PCR is shown in Figure 5, where Figure 5A is an ideal­ized version of the GEP, and Figure 5B is the actual gel. Note the distinct bands in lanes 4, 7, and 10.

A mutagenesis PCR was then run (by A. Dailey and V. Miller) using the E46K template and the K6R primers, as well as the A30P template and the K6Q/K10Q primers. The idealized GEP for this PCR is shown in Figure 6A, and the actual GEP is shown in Figure 6B. Note the distinct bands in lanes 3 and 5.

 

Figure 1: Experimental Design and Preliminary Data

(A) Diagram of the 140 codon-long α-synuclein gene, including the K6R/K10R mutations (in red), the K6Q/K10Q mutations (light green) the familial mutations, and the attached green fluorescent protein (GFP) gene. The wild-type (WT) α-synuclein is also shown. (B) 5’-3’ sequence of the forward (FP) and reverse (RP) primers for the mutagenesis PCR of α-synuclein, as well as the WT α-synuclein sequence for comparison. Nucleotide changes in the primers are highlighted in red, as are the original nucleotides in the WT sequence. The K6R primers are FP/R and RP/R, the K6R/K10R primers are FP/RR and RP/RR, and the K6Q/K10Q primers are FP/Q and RP/Q. This figure shows the sequence of every PCR primer used in this study. (C) Schematic of experimental design: α-synuclein familial mutant template plasmid and mutagenesis primers obtained (1). A mutagenesis PCR reaction was run (2). The template DNA was mutated, then methylated and destroyed, and the mutated DNA was replicated (3). The success of the mutagenesis PCR was confirmed via DNA gel electrophoresis (4), and E. coli was transformed with the mutant plasmid (5). The bacteria multiplied and the plasmid was isolated (6) and sent to the Univer­sity of Chicago for sequence confirmation (7). The plasmid was then transformed into yeast (8). (D) This gel shows the results for the primer checks. Lane 1 contains the AmpliSize ladder. Lane 2 contains the K6Q forward primer check. Lane 3 contains the K6Q reverse primer check. Lane 4 contains the K10Q forward primer check. Lane 5 contains the K10Q reverse primer check. Lane 6 contains the K6Q/ K10Q forward primer check. Lane 7 contains the K6Q/K10Q reverse primer check. Description and figure obtained from R. Thomas. (E) This gel shows the results for the primer checks. Lane 1 contains the AmpliSize ladder. Lane 2 contains the K6R forward primer check. Lane 3 contains the K6R reverse primer check. Lane 4 contains the K10R forward primer check. Lane 5 contains the K10R reverse primer check. Lane 6 contains the K6R/K10R forward primer check. Lane 7 contains the K6R/K10R reverse primer check. Description and figure obtained from R. Thomas.

 

Figure 2: Mutagenesis PCR 1 GEP

(A) Idealized version of the mutagenesis gel electrophoresis (GEP) results, showing bands that would appear if reaction was successful. Lane 1 contained a high-mass DNA ladder. 2 contained the positive control (provided by R. Thomas). 3 and 4 con­tained the mutagenesis negative control (MNC) for the K6R/K10R H50Q reaction, 5 contained the K6R/K10R H50Q mutagenesis reaction, 6 contained the K6R/K10R G51D MNC, 7 contained the K6R/K10R G51D mutagenesis reaction, 8 contained the K6R/K10R A53E MNC, and 9 contained the K6R/K10R A53E mutagenesis reac­tion. 10 contained the FP check. (B) Actual GEP. Lane content same as in A.

 

Figure 3: Mutagenesis PCR 3 GEP

(A) Idealized version of mutagenesis gel electrophoresis (GEP) results, showing bands that would appear if reaction was successful. Lane 1 contained a high-mass DNA ladder. 2 contained the mutagenesis negative control (MNC) for the K6R/K10R H50Q reaction, 3 contained a diluted mutagenesis solution for the K6R/K10R H50Q mutagenesis reaction, and 4 contained the K6R/K10R H50Q mutagenesis reaction. 5 contained the K6R/K10R G51D MNC, 6 contained the K6R/K10R G51D diluted mutagenesis reaction, and 7 contained the K6R/K10R G51D mutagenesis reaction. 8 contained the K6R/K10R A53E MNC, 9 contained the K6R/K10R A53E diluted mutagenesis reaction, and 10 contained the K6R/K10R A53E mutagenesis reaction. (B) Actual GEP. Lane contents same as in A.

 

Figure 4: Mutagenesis PCR 3 GEP

(A) Idealized version of the gel. Lanes 1 through 5 were used for K6R A53E plasmid purification confirmation (see also Figure 9), and lanes 6 through 10 were used for the mutagenesis PCR GEP of K6Q/K10Q A30P. Lanes 1 and 6 have a high-mass molecular weight ladder. Lane 2 contains the K6R A53E plasmid sample #1, 3 contains plasmid sample 2, 4 contains plasmid sample 3, and 5 contains plasmid sample 4. Lane 7 contains the MNC for K6R/K10R H50Q, and 8 contains the K6R/ K10R H50Q mutagenesis solution. Lane 9 contains the MNC for K6R/K10R E46K, and 10 contains the K6R/K10R E46K mutagenesis solution. (B) Actual GEP. Lane contents same as in A.

 

Figure 5: Mutagenesis PCR 4 GEP

(A) Idealized version of the gel. Lane 1 contains the high-mass DNA molecular weight ladder. 2 contains the K6R H50Q MNC, 3 contains the diluted K6R H50Q mutagenesis solution, and 4 contains the K6R H50Q mutagenesis solution. 5 contains the K6R G51D MNC, 6 contains the K6R G51D diluted mutagenesis, and 7 contains the K6R G51D mutagenesis solution. 8 contains the K6R A53E MNC, 9 contains the K6R A53E diluted mutagenesis, and 10 contains the K6R A53E mutagenesis solution. (B) Actual GEP, courtesy of Annika Dailey and Victoria Miller. Lane contents same as in A.

 

Figure 6: Mutagenesis PCR 5 GEP

(A) Idealized version of the gel. Lane 1 contains the high-mass DNA molecular weight ladder. 2 contains the K6R E46K MNC, and 3 contains the K6R E46K mu­tagenesis solution. 4 contains the K6Q/K10Q A30P MNC, and 5 contains the K6Q/ K10Q A30P mutagenesis solution. (B) Actual GEP, courtesy of Annika Dailey and Victoria Miller. Lane contents same as in A.

 

Bacterial Transformation

The successfully mutagenized α-synuclein plasmids were transformed into E. coli cells. First, the K6R A53E plasmid was trans­formed into E. coli cells and then spread onto transformation plates to encourage cell growth (as shown in Figure 7A-D). E. coli growth with the plasmid was observed and recorded. Transformation and mutagenesis cells were also observed for growth as controls (credit R. Thomas) (Fig­ure 7E-G).

The K6Q/K10Q A30P plasmid was also transformed into bacteria. Both the growth of the plasmid (Figure 8A-B) and the growth of control cultures (credit R. Thomas) (Figure 8C-E) were observed.

Plasmid Purification and Sequencing

To purify the K6R A53E plasmid, four E. coli colonies were iso­lated. The α-synuclein plasmid was then isolated and purified from these cells, and a GEP was run to confirm purification (idealized GEP shown in Figure 9A, actual GEP in Figure 9B). Note the distinct bands in lanes 2-5.

Four E. coli colonies with the K6Q/K10Q A30P plasmid were isolated, and the α-synuclein plasmid was purified. The idealized purification GEP is shown in Figure 10A, and the actual GEP is shown in Figure 10B.

Two samples of each plasmid type (two K6R A53E plasmids and two K6Q/K10Q A30P plasmids) were sent to UIC for sequencing. Samples of the plasmids from Figure 9 lanes 1 and 2 and Figure 10 lanes 4 and 5 were sequenced. The sequencing results are shown in Figure 11B-C whereas Figure 11A is the wild-type (WT) α-synuclein sequence.

 

Figure 7: K6R A53E Bacterial Transformation Colonies

(A) Day 1 (day of transformation: Bacterial transformation plate (with Ampicillin)): 20μL K6R A53E transformed bacteria. (B) Day 1: Bacterial transformation plate (with Ampicillin), 80μL K6R A53E transformed bacteria. (C) Day 2 (day after transforma­tion; ~7 colonies): Bacterial transformation plate (with Ampicillin), 20μL K6R A53E transformed bacteria. (D) Day 2: Bacterial transformation plate; ~25 colonies: (with Ampicillin), 80μL K6R A53E transformed bacteria. (E) Day 2: Bacterial transforma­tion control (positive control; ~4 colonies) (courtesy of R. Thomas). (F) Day 2: Bacte­rial transformation control (negative control, 0 colonies) (courtesy of R. Thomas). (G) Day 2: Bacterial transformation control (MNC; 0 colonies) (courtesy of R. Thomas).

 

Figure 8: K6Q/K10Q A30P Bacterial Transformation Colonies

(A) Day 2 (day after transformation: bacterial transformation plate (with Ampicillin)): 20μL K6Q/K10Q A30P transformed bacteria. 7 large and clearly visible colonies. (B) Day 2: Bacterial transformation plate (with Ampicillin) 80μL K6Q/K10Q A30P transformed bacteria. 14 large and clearly visible colonies. (C) Day 2: Bacterial transformation, mutagenesis positive control (courtesy of R. Thomas). 80μL bac­teria, LB+Amp. plate. (D) Day 2: Bacterial transformation, mutagenesis negative control (courtesy of R. Thomas). 80μL bacteria, LB+Amp. plate. (E) Day 2: Bacterial transformation, negative control (courtesy of R. Thomas). 80μL bacteria, LB+Amp. plate.

 

Figure 9: K6R A53E Plasmid Purification GEP

(A) Idealized version of the gel. Lanes 1 through 5 were used for K6R A53E plasmid purification confirmation, and lanes 6 through 10 were used for the mutagenesis PCR GEP of K6Q/K10Q A30P (see also Figure 4). Lanes 1 and 6 have a high-mass molecular weight ladder. Lane 2 contains the K6R A53E plasmid sample #1, 3 contains plasmid sample 2, 4 contains plasmid sample 3, and 5 contains plasmid sample 4. Lane 7 contains the MNC for K6R/K10R H50Q, and 8 contains the K6R/ K10R H50Q mutagenesis solution. Lane 9 contains the MNC for K6R/K10R E46K, and 10 contains the K6R/K10R E46K mutagenesis solution. (B) Actual GEP. Lane contents same as in A.

 

Figure 10: K6Q/K10Q A30P Plasmid Purification GEP

(A) Idealized version of the gel. Lane 1 contains the high-mass DNA molecular weight ladder. 2 contains the K6Q/K10Q A30P plasmid 1 purification solution, 3 contains plasmid 2, 4 contains plasmid 3, and 5 contains plasmid 4. (B) Actual GEP. Lane contents same as in A.

 

Figure 11: α-synuclein Mutated Plasmid Sequences

(A) α-synuclein wild-type (WT) DNA sequence, consisting of 140 codons starting at the 5’ end. (B) α-synuclein mutated with K6R and A53E. Nucleotide changed with PCR mutagenesis (K6R mutation) highlighted in red, A53E codon highlighted in red. Sequencing by UIC sequencing laboratory, obtained from R. Thomas. (C) α-synucle­in mutated with K6R/K10Q and A30P. Nucleotides changed with PCR mutagenesis (K6R/K10R mutations) highlighted in red, A30P codon highlighted in red. Sequenc­ing by UIC sequencing laboratory, obtained from R. Thomas.

 

Figure 12: K6R A53E Yeast Transformation Colonies

(A) Day 4 (3rd day after transformation): yeast transformation plate: K6R A53E, YPD plate, 30μL plasmid 1. (B) Day 4 K6R A53E, SC-URA plate, 30μL plasmid 1. (C) Day 4 K6R A53E, SC-URA plate, 200μL plasmid 1. (D) Day 4 K6R A53E, YPD plate, 30μL plasmid 2. (E) Day 4 K6R A53E, SC-URA plate, 30μL plasmid 2. (F) Day 4 K6R A53E, SC-URA plate, 200μL plasmid 2. (G) Day 4 200μL yeast, SC-URA plate. Negative control (courtesy of R. Thomas). (H) Day 4 200μL yeast, SC-URA plate. Positive control (courtesy of R. Thomas). (I) Day 4 200μL yeast, YPD plate. Neg­ative control (courtesy of R. Thomas). (J) Day 4 200μL yeast, YPD plate. Positive control (courtesy of R. Thomas).

 

Figure 13: K6Q/K10Q A30P Yeast Transformation Colonies

(A) Day 3 (2nd day after transformation): yeast transformation plate: K6Q/K10Q A30P YPD plate, 30μL plasmid 3. (B) Day 3 K6Q/K10Q A30P, SC-URA plate, 30μL plasmid 3. (C) Day 3 K6Q/K10Q A30P, SC-URA plate, 200μL plasmid 3. (D) Day 3 K6Q/K10Q A30P, YPD plate, 30μL plasmid 4. (E) Day 3 K6Q/K10Q A30P, SC-URA plate, 30μL plasmid 4. (F) Day 3 K6Q/K10Q A30P, SC-URA plate, 200μL plasmid 4. (G) Day 3 200μL yeast, SC-URA plate. Negative control (courtesy of R. Thomas). (H) Day 3 30μL yeast, SC-URA plate. Positive control (courtesy of R. Thomas). (I) Day 3 30μL yeast, YPD plate. Negative control (courtesy of R. Thomas). (J) Day 3 30μL yeast, YPD plate. Positive control (courtesy of R. Thomas).

 

Yeast Transformation

Two K6R A53E plasmid samples (the same samples with aliquots sent for sequencing) were transformed into yeast cells and spread onto transformation plates to encourage growth. Uracil deficient plates were used to ensure only the successfully transformed yeast could grow, as these cellswere transformed with a uracil-coding gene on the α-synuclein plasmid. The transformed yeast are shown in Figure 12A-F and control reactions (courtesy of R. Thomas) are shown in Figure 12G-J.

Additionally, two K6Q/K10Q A30P plasmid samples (the same samples with aliquots sent for sequencing) were then transformed into yeast cells and spread onto transformation plates. The transformed yeast are shown in Figure 13A-F, and control reactions (courtesy of R. Thomas) are shown in Figure 13G-J.

Discussion

In this study, we attempted to create multiple yeast models of α-synuclein mutations and succeeded in creating two: a K6R mutation on the familial α-synuclein mutant A53E and a K6Q/K10Q mutation on the familial α-synuclein mutant A30P. The creation of these yeast models is important because they can be utilized in future research to study how these mutations impact α-synuclein aggregation, an important factor in the study of PD.

The experiments that we performed to create these yeast mod­els were successful, from the mutagenesis PCR to the yeast transforma­tion. The sequence of the P-YES2 α-synuclein plasmid in these models was verified before we transformed it into yeast; the sequence was found to be consistent with the expected sequence ofthe mutations for each model. Although we hadsuccess with these mutant models, we failed to create the K6R/K10R mutations of the E46K, H50Q, G51D, and A53E fa­milial α-synuclein mutants, with multiple unsuccessful mutagenesis PCRs. We set out to create the K6R mutation of the familial α-synuclein mutant A53E and the K6Q/K10Q mutation of the familial α-synuclein mutant A30P because these mutations have been linked to α-synuclein aggrega­tion and PD risk. Although this link has been established, the effect of the combination of these mutations on α-synuclein aggregation is unknown. In this study, we set out to create new yeast models of these mutations to test our hypothesis and to utilize them in future research.

PCR Mutagenesis

Upon examination of Figures 2, it is evident that the K6R/ K10R H50Q, G51D, and A53E mutagenesis reactions were unsuccess­ful. Figure 2A shows the ~7 kbp (seven thousand DNA base-pair sized) bands that would have appeared on the gel in lanes 5, 7, and 9 had the reactions been successful. These bands were not present on the actual gel (Figure 2B), and thus the PCR was deemed unsuccessful. In the idealized gel, these bands show the P-YES2 plasmid (with a size of about 7 kbp) as a PCR product, which should not have appeared in the actual gel unless the reaction was succesfull.

We attempted to make the K6R/K10R H50Q, G51D, and A53E mutations again. Figure 3A shows the ~7 kbp bands that would have ap­peared in lanes 4, 7, and 10 had the reaction been successful. Figure 3B shows a lack of these bands, implying a lack of success in this reaction.

Figure 4A shows the idealized GEP for the mutagenesis of K6R/K10R H50Q and E46K, with ~7 kbp bands in lanes 8 and 10. These lanes do not appear in the actual GEP (Figure 4B) indicating the reaction was unsuccessful.

Next, the mutagenesis of K6R H50Q, G51D, and A53E was at­tempted. Figure 5A shows the idealized GEP for this PCR, with distinct ~7 kbp bands in lanes 4, 7, and 10. These bands were present in the actual GEP (Figure 5B) indicating a successful reaction.

We also attempted the mutagenesis PCR of K6Q/K10Q E46K and A30P. Figure 6A shows the idealized GEP with distinctive bands in lanes 3 and 5. These bands were present in the actual GEP (Figure 6B), indicating that the reaction was successful.

Our lack of success with the K6R/K10R attempted mutations may have occurred due to one of several reasons. Firstly, because an­other laboratory group ran mutagenesis reactions with the same primers (but different familial mutant templates) and had limited success, the issue does not rest solely on the effectiveness of these primers. More­over, our group ran the second mutagenesis PCR reaction with a different and freshly-made Master Mix solution, so this solution is also not the issue. Other groups had success with our familial templates, so those are also likely not faulty. We hypothesize that the issue may rest within the attempted combination of our K6R/K10R primers and the familial mutants we were trying to mutate. Otherpossible explanations for our lack of suc­cess could rest in the quality of the polymerases used in the master mix, or additionalfactor we have not considered (Munteanu, Braun, & Boonrod, 2012).

Bacterial Transformation and Plasmid Purification

We expected to transform and purify the plasmids with the K6R/K10R mutations; however, due to a lack of mutagenesis success, we did not attempt to transform them into E. coli cells or purify the plasmids. With the K6R and K6Q/K10Q mutations on their respective templates, we set out to transform them into E. coli and purify their plasmids which we were able to do successfully. Our E. coli transformation occurred with no issues. Figure 7 shows the transformed colonies for K6R A53E. Since these colonies were grown on an ampicillin-rich medium, the only bacteria that could grow were those that had been transformed properly, resulting in the expression of a protein conferring ampicillin resistance to these bacteria. Figure 8 shows the successful transformation of the K6Q/K10Q A30P plasmid into bacteria. We successfully isolated multiple colonies on both plates (with 20μL and 80μL of E. coli cells spread on them). On the 80μL plate, we had about three times as many individual colonies as the 20μL plate, which indicated that higher levels of bacterial solution are correlated with higher numbers of bacterial colonies.

We next set out to purify our α-synuclein plasmids from E. coli cells and expected the sequence confirmation to show that the plasmids we were working with were indeed mutagenized correctly. We observed successful purification and sequencing with our K6R and K6Q/K10Q mutations. The purification of the plasmid from four K6R A53E bacterial colonies was confirmed via GEP (Figure 9). In this figure, the presence of distinctive bands in lanes 2, 3, 4, and 5 shows the successful purification of all four plasmid samples. Plasmids 1 and 2 (from lanes 2 and 3 respec­tively) were sequenced, and their sequences were as expected given the mutations of the plasmid (Figure 11B). The purification of the K6Q/K10Q A30P plasmid from four bacterial colonies was confirmed via GEP (Figure 10). In this GEP, lane 2 (plasmid 1) did not show a distinctive band, indicating that this purification was unsuccessful (this was not completely unexpected given visual differences in this sample before and during the purification protocol). Lane 3 (plasmid 2) shows a weak purification band, indicating that the purification may have only been partially successful. Lanes 4 and 5 (plasmids 3 and 4 respectively) showed distinctive bands, indicating that the purification was successful. As a result, plasmids 3 and 4 were sequenced, and the obtained sequences were shown to be as expected given the mutations of the plasmid (Figure 11C).

Yeast Transformation

Our K6R A53E and K6Q/Κ10Q A30P plasmids were then transformed into yeast, which proliferated as expected (Figures 12 and 13, respectively). Our success came from not only our correctly purified and sequenced plasmids, but also from the conditions on which the yeast were allowed to grow. By having optimal growth media and incubation temperature, our yeast expressing the mutated α-synuclein protein pro­liferated as expected. The yeast were grown on a uracil-deficient media (SC-URA) and as a result, only the yeast that were transformed properly (which enables them to produce uracil) could proliferate.

Future of the Yeast Models

In future research, these yeast models can be used to examine α-synuclein aggregation and its impacts on PD. For example, one could examine the impact of these mutations on the growth of yeast cells using a spotting assay (Duennwald, 2012). One could also perform immunos­taining with an α-synuclein antibody and examine the distribution and ac­cumulation of α-synuclein in these mutant models (Zabrocki et al, 2005). One could also examine how mutant α-synuclein interacts with other proteins in yeast. We hypothesize that the K6R A53E yeast model would show a lower rate of growth, as well as increased α-synuclein aggrega­tion. The K6Q/K10Q A30P model would likely show decreased aggrega­tion and a higher growth rate compared to K6R A53E, as the K6Q/K10Q mutations have been connected to decreased aggregation.

Additionally, phosphorylation of α-synuclein on serine 129 is a major modification found in Lewy bodies. PLK2 phosphorylates α-synu­clein on serine 129 and increases α-synuclein cytotoxicity within yeast (Basso et al, 2013). This amino acid-specific modification would be inter­esting to study within our own yeast models to examine how PLK2 would interact with our mutant yeast. Our yeast model may show interactions between PLK2 and serine 129 not seen in other forms of α-synuclein. If one of our α-synuclein models is resistant to an increase of cytotoxicity normally caused by PLK2, then our α-synuclein model might be linked to less Lewy body aggregation.

Proteasomal dysfunction and oxidative stress diversely impact α-synuclein localization and aggregation, and both impairments induce cellular toxicity (Sharma et al, 2006). Examining the effects proteasomal dysfunction and oxidative stress have on our α-synuclein yeast models would offer insight into how these complications affect α-synuclein mu­tant’s ability to aggregate into Lewy bodies and its link to PD in general.

In addition, α-synuclein toxicity increases as a result of other mechanisms acting upon it. The endosomal-lysosomal pathway links α-synuclein and the VPS35 protein in yeast, which has been shown to enhance α-synuclein aggregation within yeast and is involved in the pathogenesis of PD (Huang et al, 2017). Studying VPS35’s α-synuclein aggregation enhancement on our own α-synuclein models may reveal aggregation properties and alternatives for α-synuclein to react in the endosomal-lysosomal pathway. Studying α-synuclein cytotoxicity and its enhancement of aggregation provides insight of some of the mechanisms that cause Lewy body aggregation. These mechanisms explain why and how α-synuclein aggregation might be occurring but fail to address how α-synuclein aggregation could be prevented or reversed. Nicotine has been found to slow down aggregation of α-synuclein and even purify the cytotoxicity in an α-synuclein yeast model of PD (Kardani, Sethi, and Roy, 2017). Examining the effects nicotine would have on our α-synuclein mu­tant yeast model could produce an even greater reduction in α-synuclein aggregation or even complete extinction of the property.

In our experiment, we created two mutant yeast models of α-sy­nuclein thatcan be used in future PD research. These models will lead to a greater understanding of how mutations impact α-synuclein aggrega­tion into Lewy bodies andas PD in general. Our yeast models will allow researchers to observe the properties of our mutations of α-synuclein and can shed light into how α-synuclein aggregation can be regulated; this may lead to potential new treatments for the disease.

Methods

Primer Design and Synthesis

To create our forward and reverse primers (Figure 1B), as stat­ed in the lab manual (DebBurman 21-22), we looked at the 140 codon, 420 nucleotide-long α-synuclein coding sequence and then proceeded to determine which amino acids would be manipulated to make our muta­tion. First, we designed the primers for K6R/K10R. To create the forward primer for this mutation, we changed the AAA (Lysine) nucleotide codon sequence on the 6th codon position to the nucleotide sequence of AGA, which codes for the amino acid Argenine. We also changed the AAG nucleotide sequence (which also codes for Lysine) on the 10th codon to AGG, which also codes for the amino acid Argenine. The K6R forward primer was designed with the same sequence as the K6R/K10R primer but without the codon changes resulting in the K10R mutation. The K6Q/ K10Q primer was designed with a change on the 6th codon from AAA to CAA (Glutamine) and a change on the 10th codon from AAG to CAG (which also codes for Glutamine). All primers used extended from the 1st to the 16th codon of α-synuclein.

Reverse primers were also designed and had the same length as the forward primers. The reverse primers were designed using Wat­son-Crick base-pairing rules as complementary to their respective forward primers but in the 5’ to 3’ direction (the same direction as the forward primers). Thus, when a reverse primer is read, the first codon is the 16th codon in the forward primer but read in the opposite direction. The sec­ond codon in the reverse primer is the 15th in the forward primer, and this continues until the sixteenth codon (1st in the forward primer). This was our mutagenesis primer design; Figure 1B shows the primer sequences used in our experiments.

Template DNA, Vectors, Bacterial and Yeast Cells

The template DNA that we used in this experiment is called a vector, which is a circular, double-stranded piece of DNA. The unique property of this template DNA is that it is not attached to chromosomal DNA, so it is capable of replicating on its own. This makes it ideal for our experiment as we can mutate it and then insert it into E. Coli, where it will be able to replicate (DebBurman 35). The vector that we used was the PYES2 plasmid vector. The bacterial cells that we used were E. Coli. Be­cause bacteria replicate quickly and plasmid vectors can be transformed into them, they are ideal for assisting researchers in quickly replicating many copies of a plasmid vector. Each cell doubles every 20 minutes; and as a result, colonies are formed rapidly. We used E Coli as a vehicle of replication for our K6R A53E α-synuclein mutation in the PYES2 plasmid vector. The yeast that we used was budding yeast (S. cerevisi­ae). Yeast is a good model organism because it behaves very similarly to a human cell and allows for knockout of specific genes (Kain, 2008). It has also been shown to be an effective and useful model of α-synuclein aggregation and toxicity in PD (Fiske et al., 2011).

Plasmid-based PCR Mutagenesis

To create our mutation of the wild-type α-synuclein gene in the plasmid vector DNA, we used a technique called a polymerase chain reaction (PCR). We performed it according to the steps in the lab manual (DebBurman 24-27). To prepare the mutagenesis reaction, we combined the following:

32.6 μL Sterile H2O

1.5 μL Forward primer

1.5 μL Reverse primer

1 μL Purified plasmid

13.4 μL Mutagenesis master mix

(for our negative control, we used no master mix and 46 μL of water to compensate for the lack of master mix)

We also created a forward primer control check using the same steps as above to demonstrate that the primers bind to template DNA. To prepare this reaction the following amounts of each reaction additive were used:

22.5 μL H2O

1.5 μL Forward primer

1.5 μL Reverse primer

1 μL purified plasmid

25 μL Mutagenesis master mix

We then ran the PCR reaction by running the samples in the PCR machines at the temperatures and times provided in the lab manual (DebBurman 26).

Our peer teacher ran a mutagenesis positive control reaction for us by using pUC19WHITE control plasmid in place of our purified plasmid and 10X Accuprime pfx reaction mix, 10X enhancer, DNA meth­ylase, 25X SAM, and Accuprime pfx. This reaction was run according to the same PCR method as with the reactions above, described in the lab manual.

Recombination and E. coli transformation

The next step was to perform a recombination reaction and do bacterial transformation. We did this according to the lab manual (Deb­Burman Pg. 32). We did the recombination to enhance the efficiency of the mutagenesis and increase colony yield; we transformed the plasmids into bacteria so that they could replicate quickly and produce high yield of mutated plasmids. The materials that we used for this reaction were as follows:

Competent DH5 alpha Bacteria cells

PCR water

Mutagenesis PCR sample

SOC media

2X enzyme mix

0.5 M EDTA

We followed the steps in the lab manual for the recombination reaction and then proceeded to perform the steps for the bacterial transformation. After we completed the bacterial transformation, we incubated the plated bacteria in a 37-degree Celsius environment for overnight growth.

Plasmid Purification and Gel Electrophoresis

In this part of the experiment, we purified the pYES2 plasmid vectors from the bacteria according to the lab manual (DebBurman 35- 38). We wanted to increase our chances of high amplification yield of our α-synuclein mutation in the plasmid vectors and eliminate the cells that still contain plasmid vectors that are not mutated for α-synuclein, so we performed plasmid purification of four E. Coli colonies. To remove the plasmids from the E. Coli without damaging it, an alkaline method of puri­fication was used. This method involved isolating the plasmid from E. Coli that had been grown on a LB plate and allowed to replicate. The lysate was first removed from the bacteria, the DNA was absorbed through our QIA prep membrane, and the plasmids were then washed and eluted.

The plasmid was then checked with gel electrophoresis. First, we prepared the gel as described in the lab manual, then we created the mixture that we were going to run in the gel using the following:

5 μL Purified plasmid

7.5 μL Sterile H2O

1.5 μL 10X loading buffer

These samples were added to the gel, along with a 2000 bp molecu­lar weight ladder. The gel was ran, and the results were imaged by Dr. Wilcox.

DNA Sequencing

DNA sequencing of our mutated plasmid was performed at the University of Chicago sequencing and genotype facility. It was performed according to a method called “the chain termination method”. Our plasmids were sent with a sequencing primer: the gal forward primer. The chain termination method involves combining the DNA in a tube with the primer, DNA polymerase, and DNA nucleotides (dATP, dTTP, dGTP, and dCTP). They heated the mixture to denature the DNA primer and then cooled it so that the primer could bind to the template. After this, the temperature is raised again, which makes it so that the polymerase could synthesize new DNA. After this was complete, the DNA was analyzed using gel electrophoresis (Obenrder 1-2).

Yeast transformation

The final step in our experiment was to transform our mutated α-synuclein, contained within the PYES2 plasmid vector, into budding yeast. This process was followed as stated in the lab manual (DebBur­man 39-41). To transform the mutated α-synuclein into yeast, we had to increase the permeability of its cell wall, as cell walls make insertion of plasmid tricky. To do this, we used a technique called LiAc transformation.

First, the mutated α-synuclein was cultured to produce a sufficient amount for the experiment (this step was performed by the lab teacher). Then, the plasmids were transformed into yeast. The transformation mixture used in this part of the experiment was:

240 μL of PEG (50% w/v)

36 μL of 1.0 M LiAc

25 μL of single-stranded carrier DNA (2.0mg/mL)

46 μL of H2O

Plasmid DNA (.5-10 μL)

After the steps to transform the PYES2 plasmid vector containing the K6R/K10R A30P mutation were completed, the samples were then plated onto three plates per sample of each of the following:

20 μL on one Sc-Ura glucose

200 μL on one Sc-Ura glucose

20 μL on YPD

After plating, the samples were incubated and the yeast colonies were allowed to grow for five days. They were then stored in the freezer.

Author Contributions

What follows were the contributions made to this paper by each author. A.W. and S.K.D. conceptualized and designed experiments. A.W. supervised. P.F., L.P., and A.S. wrote the initial paper and preformed ex­periments. P.F. made figures and formatted the paper. P.F. and L.P. wrote the final draft of the paper.

Acknowledgements

We would like to thank the multiple people who assisted and guided us during the writing process.First, we would like to thank Dr. DebBurman (BIOL 221 professor) for kindly letting us assist him in his re­search on the role of a-synuclein in PD and for his guidance and support in this research. We would like to thank Dr. Wilcox (BIOL 221 laboratory professor) for his patient guidance and instruction in the laboratory and for his invaluable feedback and mentorship in all of our projects. Thanks to Annika Dailey and Victoria Miller for conducting the K6R A53E and K6Q/K10Q A30P mutagenesis reactions. All mutagenesis data for these mutations was obtained by Annika and Victoria. We would like to thank Rosemary Thomas (BIOL 221 laboratory peer teacher) for guiding us in the laboratory, demonstrating how to properly perform experimental protocols, re-doing parts of our experiments that were unsuccessful, preparing laboratory materials for our use, suggesting edits for our paper, and for always being of assistance and support. Finally, we would like to thank Lake Forest College for providing us with the opportunity and all of the necessary tools to perform this important research on the role of α-synuclein in Parkinson’s Disease.

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