Summary

Parkinson’s disease is a hypokinetic movement disorder marked by the loss of dopaminergic neurons in the midbrain. Its main pathological marker is the accumulation of Lewy bodies, which are made up of misfolded alpha-synuclein. In addition to the six known familial mutations, posttranslational mutations like glycation and acetylation have also been shown to influence alpha-synucle­in’s toxicity. To try and investigate the effects these mutations and modifications have on alpha-synuclein, we attempted to make a K6Q mutation on A30P, A53T, and a K6Q/K10Q mutation on G51D. These mutants were created by a step- by- step mutagenesis reaction. We hypothesized that all mutations would be created, successfully ac­cepted into E. coli for growth, and would be transformed into yeast to be prepared for further assays. The overall predictions for the success of these mutations were: 1) the K6Q mutation on A30P and A53T would result in less aggregation of familial mutant alpha-synu­clein, which leads to its toxicity and results in genetic Parkinson’s Disease, and 2) the K6Q/K10Q mutation on G51D would further exacerbate these effects. All mutations were successfully created, taken in by E. coli to grow more copies of the mutated plasmid, and transformed into yeast. Important knowledge has been gained from this experiment, but we are still far from finished. Only further assays will prove whether progress was truly made in the vastly uncertain world of Parkinson’s Disease.

Introduction

Parkinson’s Disease (PD) is characterized by the loss of dopaminergic neurons in the midbrain. It is the second most common neurodegenerative disease after Alzheimer’s (Ross, Braithwaite, Farrer, 2008). There are currently more than four million cases of PD worldwide; the diagnosis is one that a general physician can make (Outeiro, 2003). Parkinson’s Disease is a hypokinetic movement disorder: “hypo” meaning less, “kinetic” meaning movement. The requirements for a diagnosis of Parkinson’s Disease is any combination of the following movement abnormalities: tremor-at-rest, bradykinesia, poor posture, rigidity, shuffling gait, poor balance, and the “freezing phenomenon” (Fahn, 2008). The di­agnosis, however, does require an individual to present with bradykinesia and tremor-at-rest as defining symptoms (Fahn, 2008). These symptoms do not necessarily lead to death in patients, but a secondary cause stem­ming from these symptoms may lead to mortality (Fahn, 2008).

Parkinson’s Disease and several other neurodegenerative diseases all fall under a broader category of diseases known as synucle­inopathies, in which the protein alpha-synuclein forms clumps (aggre­gates) and kills the cells (Dawson & Dawson, 2003). The key pathological marker that defines PD is the presence of Lewy bodies on the inside of neuronal cells. Lewy bodies are mainly composed of misfolded α-sy­nuclein protein (Petrucelli & Dickson, 2008). There are two main types of Parkinson’s Disease: sporadic and familial. 90% of the cases are sporadic, but it has been difficult to identify the causes of development for the sporadic forms. Parkinson’s Disease may be described as a neurode­generative disease with many possible etiologies. Possible etiologies of sporadic-type PD that have been suggested are altered metal homeosta­sis, environmental toxins, and mitochondrial dysfunction (Bossy-Wetzel et al., 2004). Certain gene mutations in α-synuclein have been discovered to play a role in the development of genetic, or familial, Parkinson’s Disease (Ross et al., 2008). When dopaminergic neurons are functioning properly, they give the substantia nigra in the midbrain bands of a characteristic dark pigment. The aggregation of α-synuclein protein in the dopaminergic regions of the substantia nigra results in a noticeable depigmentation in these regions. About 90% of these dopaminergic neurons die before the first symptoms are even present (Petrucelli and Dickson, 2008). There are many genes that can lead to the onset of Parkinson’s disease other than alpha-synuclein. Some disease-causing genes include Parkin, Pink1, DJ-1, ATP13, LRRK2, and VPS35. Some examples of risk factor genes include SAC1, VPS13, and SWA2. (Brás, Guerreiro, and Hardy, 2015). In addition, there are six familial mutations on alpha synuclein that are directly linked to genetic Parkinson’s Disease that will be described later in this article.

The function of α-synuclein remains mostly unclear. It is one of the most abundant proteins in the brain, and it works at the synapse level. Due to its abundance in presynaptic terminals and synaptic vesicles, there is evidence that α-synuclein plays a key role in vesicle transport and neurotransmission (Abeliovich et al, 2000). In mice, it has been shown that knocking out α-synuclein leads to a decrease of synaptic vesicles, synaptic responses upon stimulation, and a poorer recovery of synaptic strength from use-dependent depression (Chandra et al., 2004). One may argue that this is evidence suggesting that α-synuclein could be involved in brain plasticity, a phenomenon that invertebrates do not experience (Szego et al., 2012). Alpha synuclein is 140 amino acids long and has three domains, each with corresponding functions: membrane binding in the N domain, aggregation in the M domain, and solubility in the C domain (Bartels et. al, 2010). There are six familial mutations - implicated in PD that are concentrated in the N-domain, and posttranslational modifi­cation sites for glycation and acetylation have been found there. There is a common hypothesis that Parkinson’s Disease is a result of α-synuclein coming out of solution, misfolding, and aggregating, leading to its cellular toxicity. The influence of posttranslational modifications and the presence of a familial mutation may affect the localization, accumulation, or solubili­ty of the protein.

As mentioned previously, there are six mutations known to cause familial Parkinson’s disease, and much more is known about these 10% of PD cases. The six known familial gene mutations found in the N-terminus of α-synuclein are: A30P, E46K, H50Q, G51D, A53T, and A53E. A53T, E46K, H50Q and the wild-type α-synuclein mutations have an increased affinity for lipid membrane binding (Outeiro, 2003). This results in toxic levels of aggregation and what we know as Lewy Bodies. In addition, in the A30P and A53T mutants, the catabolic pathway by which wild-type α-synuclein is normally degraded is affected, which leads to further accumulation and toxicity (Ghosh et al., 2014). In the A30P and G51D mutants, protein aggregates are mainly cytoplasmic as they exhibit impaired membrane association. This is most likely due to a defect in endocytosis. In fact, these two mutations behave very similarly in yeast (Fares et al., 2014). One defining characteristic of G51D is that it enhanc­es mitochondrial fragmentation in primary neurons. It is believed that this is where its toxicity lies, given that it does not bind to membranes. H50Q produces toxicity by enhancing aggregation of α-synuclein in neurons (Fares et al., 2014). The A53E mutation has a mechanism that is yet to be delineated; however, it does show reduced aggregation in cells, indicating that, instead of generating large amounts of aggregation, the mutation has a different mechanism of toxicity (Rutherford and Giasson, 2015). Individuals who have any of the six known mutations will develop early-onset Parkinson’s Disease.

Glycation and acetylation are two post-translational modifica­tions that can occur in proteins. Glycation isan age-related post-transla­tional modification that has beenshown to enhance α-synuclein toxicity in Drosophila and mice (Miranda et al., 2017). Glycation influences the N-terminus of α-synuclein and has been shown to reduce membrane binding and impair its clearance (Miranda et al., 2017). In other words, glycation within the protein α-synuclein produces negative effects on the cells. Research has shown that α-synuclein can be acetylated and glycat­ed on lysines 6 and 10, and that these sites can be deacetylated by the protein sirtuin 2 (De Oliveira et al., 2017). A mutation- blocking acetylation in the substantia nigra in rats has been shown to decrease α-synuclein toxicity. This goes to show that acetylation may be a regulatory mecha­nism when it comes to the aggregation and toxicity of α-synuclein (De Oliveira et al., 2017). Acetylation has been shown to have a positive influ­ence on cells while glycation has a negative impact. While some research has been done on both post-translational modifications, the mechanisms of their interaction with the six known familial mutations on α-synuclein are unknown.

The most common and appropriate organism in which to test these mutations and others is budding yeast, or Saccharomyces cerevisi­ae. Budding yeast is a single-celled eukaryote, so it contains membrane-bound organelles likehuman cells (Duina et al., 2014). The genome of yeast has been completely sequenced and described because of its small size, not to mention that the genes and proteins in yeast are very similar in function to those in humans. Saccharomyces cerevisiae reproduces quite readily and very quickly by “budding”, which allows scientists to conduct research (Allendoerfer, 2008). The key feature that diversifies budding yeast as a model organism is the simplicity of adding or remov­ing genes from the cell, whether it be by means of a plasmid that has been replicated in bacteria beforehand, or by inserting new genes directly into the chromosomes (Duina et al., 2014). Since the important pathology of Parkinson’s Disease involves the misfolding of a protein, budding yeast is an excellent model organism to study it because they make, fold, and degrade proteins just like humans do (Sharma et. al, 2006).

To establish the interaction of glycation and acetylation and the six known familial mutations, we first plan to create a K6Q mutation in which lysine is being converted to a glutamine on the 6th amino acid. We will then combine this mutation with the six known familial mutations. We will also create a K6Q/K10Q mutation in which lysine is being converted to a glutamate on the 6th and 10th amino acids and combine this muta­tion with the G51D familial mutation. These mutations will block glycation and mimic acetylation on these particular sites. In doing this, we will be ultimately creating seven mutagenized products. To gain success, we have four aims: 1) create the mutants by PCR mutagenesis, 2) transform them into bacteria,3) purify the plasmid from the bacteria and sequence it, and 4) transform the mutants into S. cerevisiae for future assays. For the purposes of the K6Q mutation, we hypothesize that the mutation will be successfully created, accepted by both E. coli and yeast, and will be phe­notypically different from wild-type α-synuclein. We hypothesize that com­bining the K6Q or the K6Q/K10Q mutation with the six familial mutants will exacerbate the phenotype elicited by the K6Q or K6Q/K610 mutation alone because the familial mutations already have a negative impact on cells. Our hypothesis regarding the mutation is that the acetylation mutation on α-synuclein will be different in the way that it folds, which will cause it to be less “sticky than wild-type and familial mutant α-synuclein. By acetylating amino acid 6 and/or 10, this will result in steric hindrance due to the large acetyl group and will therefore prevent misfolding of the protein (Iyer et al., 2016). The normal “misfolding” will not be able to take place due to the conformational difficulty, preventing unfavorable folding and protein aggregation in the nerve cells of the brain.

Results

Primer Design and K6Q Mutagenesis of A30P, A53T, and E46K Alpha-Sy­nuclein and K6Q/K10Q Mutagenesis of G51D Alpha-Synuclein

An alpha-synuclein cartoon in the Wild Type (WT) version was compared to our mutant plasmids in Figure 1A. The goal of the project was to create a K6Q mutation with the familial mutations A30P, E46K, and A53T, and a K6Q/K10Q mutation with the familial mutation G51D. The location of the amino acid mutation is indicated in Figure 1A. The primers for the mutations and WT sequences are depicted in Figure 1B. The over­all schematic of our experimental design is shown in Figure 1C. The goal was to create our mutants in Saccharomyces cerevisiae in a PYES2 DNA vector. To do this, we performed a PCR reaction using full length alpha-synuclein in addition to the crafted primers. When proven successful, we transformed the mutagenized alpha synuclein into the bacteria, E. coli. This destroyed the unwanted template DNA. After the mutant plasmid was isolated, we purified it from the E. coli and confirmed the results using gel electrophoresis. When the gel electrophoresis indicated that the plasmid was present, we sent it to the University of Chicago for sequenc­ing. When the sequence indicated that we had successfully mutagenized our desired mutants, we transformed them into yeast to perform future assays. Before beginning the project, our peer leader Chisomo Mwale ran a primer check using wild type alpha synuclein on the forward and reverse primers for the K6Q mutation and the K6Q/K10Q mutation using WT alpha-synuclein by running a gel electrophoresis. The results are shown in Figure 1D. Primers were designed based on the guidance of DebBurman, 19-21.

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Figure 1. Creating the Mutations and the Overall Schematic

(A) Alpha-synuclein cartoon (WT) vs. Mutant plasmids K6Q A30P, K6Q A53T, and K6Q E46K, including the GFP gene. We predict that mimicking acetylation by means of the K6Q mutation, the proteins containing the familial mutations will aggregate and “stick” less. Top right: A30P-familial mutation: Amino acid 30 is changed from alanine to proline. K6Q mutation is done on alpha-synuclein with the A30P mutation. K6Q: Lysine to glutamine. Bottom left: E46K-familial mutation: Amino acid 46 is changed from glutamic acid to lysine. K6Q mutation is done on alpha-synuclein with the E46K mutation. K6Q: Lysine to glutamine. Bottom right: A53T-familial mutation: Amino acid 53 is changed from alanine to threonine. K6Q mutation is done on alpha-synuclein with the A53T mutation. K6Q: Lysine to glutamine.

(B) PCR primer pairs for each mutation and for control reactions.

(C) Overall schematic of experimental design. Step 1: create primers and synthesize by PCR mutagenesis. Step 2: Transform into bacteria. Step 3: Amplify mutant DNA in bacteria. Step 4: Isolate mutant plasmid by plasmid purification. Step 5: Send to university of Chicago for sequencing. Step 6: Transform into yeast.

(D) This gel shows the results for the primer checks. All success is displayed by the presence of dark bands in lanes 2-7. In lane 2, The K6Q forward primer check was successful. In lane 3, the K6Q reverse primer check was successful. In lane 4, the K10Q forward primer check was successful. In lane 5, the K10Q reverse primer check was a success. Lane 6, which contained the K6Q/K10Q forward primer check and lane 7, which contained the K6Q/K10Q reverse primer check were also com­pleted successfully, identifiable by the dark bands between .8 and 1.5 kb.

K6Q A30P, K6Q E46K, K6Q A53T and K6Q/K10Q G51D Gel Electropho­resis

Using the forward and reverse primers for K6Q A30P, K6Q E46K, K6Q A53T and K6Q/K10Q G51D, mutagenesis reactions were done following the procedure in DebBurman, 28-34. Control reactions for the primers (positive and negative) were also created. This procedure included running a Polymerase Chain Reaction (PCR). A DNA Agarose gel was run to separate the PCR products. The ideal versions of the gels are shown in Figures 2A-2C and they indicate where a dark black band is expected to be. Results of the actual DNA gel are shown in Figures 2D-F. Successful mutations can be determined by comparing the ideal gel and the actual gel; if the bands are in the same location, they can be deemed successful.

K6Q A30P, K6Q A53T and K6Q/K10Q G51D Bacterial Transformations

The next step was to transform the PCR products for K6Q A30P, K6Q E46K, K6Q A53T and K6Q/K10Q G51D into E. coli follow­ing the procedure in DebBurman 28-34. This was done to get rid of the original template vector and keep only the mutagenized product as well as replicate them. Lysogeny Broth (E. coli) + Ampicillin (LB+ Amp) Agar plates were used to allow only cells that contained a vector with Ampicillin resistance to grow. Both 20μl and 80μl of cells were plated onto the LB+ Amp plates. Chisomo Mwale created positive and negative controls for the class, shown in Figure 3A. These plates show a lot of growth for the positive control and no growth on the negative controls. Figure 3B shows the transformation of K6Q A30P. On both the 20μl and 80μl E. coli + Amp plates the white dots indicate the presence of bacterial colonies. Figure 3C shows the transformation of K6Q A53T. Few colonies are present on both the 20μl and 80μl plates. Finally, Figure 3D shows transformation of K6Q/K10Q G51D into E. coli.

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Figures A and D Legend:

Lane 10: DNA Ladder (10Kb)

Lane 9: Mutagenesis Positive Control.

Lane 8: Familial Mutant A30P Negative Control.

Lane 7: Familial mutant A30P K6Q mutagenesis.

Lane 6: Familial Mutant E46K Negative Control.

Lane 5: Familial Mutant E46K K6Q mutagenesis.

Lane 4: Familial Mutant A53T Negative Control.

Lane 3: Familial Mutant A53T K6Q mutagenesis.

Lane 2: PCR Forward Primer Check Negative Control.

Lane 1: PCR Forward Primer Check.

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Figures B and E Legend:

Lane 1: nothing.

Lane 2: K6Q –A30P.

Lane 3: K6Q +A30P.

Lane 4: K6Q/K10Q -A30P.

Lane 5: K6Q/K10Q +A30P.

Lane 6: K6Q/K10Q -A53T.

Lane 7: K6Q/K10Q +A53T.

Lane 8: K10R –A53T.

Lane 9: K10R +A53T.

Lane 10: MW Ladder.

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Figures C and F Legend:

Lane 1: nothing.

Lane 2: High Mass Ladder.

Lane 3: A30P Background.

Lane 4: K6Q/K10Q/A30P.

Lane 5: E46K Background.

Lane 6: K6Q/K10Q /E46K.

Lane 7: A53T Background.

Lane 8: K6Q/K10Q /A53T.

Lane 9: H50Q Background.

Lane 10: K6Q/K10Q /H50Q.

Lane 11: G51D Background.

Lane 12: K6Q/K10Q/G51D.

Lane 13: A53E Background.

Lane 14: K6Q/K10Q /A53E

Figure 2. PCR Mutagenesis Gels

(A) This gel shows the ideal gel for the K6Q mutagenesis of A30P, E46K, A53T, and PCR FP check. Gel was run backwards. See figure legend.

(B) This gel shows the Ideal gel for the mutagenesis of K6Q A30P, K6Q/K10Q A30P, K6Q/K10Q A53t, and K10R A53T. This gel was run backwards. See figure legend.

(C) This gel shows the ideal gel for the K6Q/K10Q mutagenesis of A30P, E46K, A53T, H50Q, and G51D. See figure legend.

(D) This gel shows the results for mutagenesis of A30P, E46K, A53T, and PCR FP check. See figure legend. The presence of a band at approximately 6kb is indicative of a successful PCR product.

(E) This gel shows the mutagenesis of K6Q A30P, K6Q/K10Q A30P, K6Q/K10Q A53T, and K10R A53T. This gel was run backwards. See figure legend. A relatively dark band is present at about 6kb which matches our K6Q A30P mutation on the ideal gel.

(F) This gel shows the K6Q/K10Q mutagenesis of A30P, E46K, A53T, H50Q, and G51D. There is a relatively faint band in lane 12, but it is dark enough to transform into bacteria.

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