Creation of Novel alpha-Synuclein Mutation A53E Truncations 123 and 120 for Future Study in Budding Yeast

Parkinson’s disease (PD) is a neurological disorder resulting from the loss of dopaminergic neurons in the substantia nigra. ɑ-Synuclein, a PD-associated protein, clumps and forms aggregates, or Lewy bodies, in PD patients. Recently, a novel familial mutation of ɑ-synuclein, A53E, was discovered. Our study aimed to create two truncated versions of the familial mutant A53E by removing amino acids from the C-terminus of the protein in order to use to analyze in future studies compared to the more well studied wild-type and full-length versions of ɑ-synuclein. We hypothesized that the A53E mutation, as well as the C-terminus truncations, would cause a more aggressive pathology than wild-type or full-length ɑ-synuclein. As more amino acids were removed from the C-terminus of ɑ-synuclein, we predicted solubility to decrease and aggregation to increase. We successfully PCR amplified truncations A53E 123 and 120, displaying correct protein length, later subcloning and transforming the purified fragments into E. coli. Bacterial transformation produced positive colony growth, which was used for transformation into S. cerevisiae. The two truncations of ɑ-synuclein created, A53E 123 and 120, will be used in future studies to study the properties ɑ-synuclein displays in yeast, including patterns in localization and toxicity. Introduction Parkinson’s disease (PD) is a progressive movement disorder characterized by neuronal loss and Lewy body formation (Spillantini et al., 1997; Polymeropoulos et al., 1997). Symptoms include tremor-at-rest, bradykinesia, and stiffness of limbs (Fahn, 2008). Parkinson’s disease is characterized by a loss of dopaminergic neurons in the substantia nigra (Polymeropoulos et al., 1997). In the diminished substantia nigra of PD patients, clumps of protein, called Lewy bodies, are present (Zabrocki et al., 2005). Lewy bodies are formed from the naturally occurring and abundant protein ɑ-synuclein. ɑ-Synuclein serves to release chemicals in the brain, has an unusually flexible shape, and is characterized by two main features: it likes to bind to membranes and form aggregates (Jin et al., 2011). In those with PD, too much ɑ-synuclein is being made or too little is being degraded, creating an imbalance. This imbalance is generated when ɑ-synuclein misfolds and accumulates to form Lewy bodies, leading to toxicity and neuron death (Petrucelli and Dickson, 2008; Jin et al., 2011). There are two types of Parkinson’s disease, sporadic, accounting for 90 percent of individuals, and familial, accounting for 10 percent of individuals (Fahn, 2008). These two types are used to differentiate genetic from idiopathic forms of PD. There are eight known genes that, when mutant, can lead to genetic forms of PD. The first, SNCA, is the gene that encodes the protein ɑ-synuclein, the central problem in both genetic and sporadic PD. The other seven: Parkin, UCH-L1, PINK1, DJ-1, LRRK2, ATP13A2, and HTRA2, can also misfold ɑ-synuclein to form Lewy bodies (Ross, 2008; Polymeropoulos et al., 1997). Past research has shown ɑ-synuclein can be mutated, modified by phosphates and nitrates, and shortened into smaller versions (Polymeropoulos et al., 1997; Fujiwara et al., 2002). Our research will explore mutated and smaller versions of ɑ-synuclein. We will focus on a recently discovered familial mutation of ɑ-synuclein, A53E, and how cutting this mutation into smaller versions will affect the membrane binding, aggregation, and solubility of ɑ-synuclein. Two truncated forms of ɑ-synuclein, 123 and 120, will be designed in this study. Truncation occurs by removing amino acids from the C-terminus of ɑ-synuclein, making the protein shorter. Truncations at the carboxyl and amino termini are naturally occurring in Lewy bodies, which are characteristic of PD. The C-terminus is crucial for maintaining the shape of ɑ-synuclein, which keeps the protein soluble (Hong et al., 2011). The C-terminus is rich in glutamic and aspartic residues similar to proteins with chaperone-like activity. It was found that C-terminal truncated ɑ-synuclein does not possess chaperone-like activity, showing the C-terminus plays a role in this activity and is important for maintaining ɑ-synuclein solubility as C-terminal truncated ɑ-synuclein aggregates faster than fulllength ɑ-synuclein (Souza et al., 2000). Additionally, Kanda et al. (2000) found truncations at the 120 amino acid in the C-terminus are more susceptible to oxidative stress, which is hypothesized to influence neuronal loss in PD patients. Several mutations in ɑ-synuclein have been found to cause genetic forms of PD, including A53T, E46K, A30P, H50Q, and G51D. Kanda et al. (2000) found the two well-studied familial mutations A53T and A30P are affected by oxidative stress, causing neuronal cell death associated with PD. A53T is known to cause ɑ-synuclein to bind to the membrane, while A30P localizes ɑ-synuclein in the cytoplasm (Sharma et al., 2006). A53T is more aggressive, suggesting that toxicity involves ɑ-synuclein binding to the membrane (Kanda et al., 2000; Sharma et al., 2006). Another familial mutant, E46K, also binds to the membrane, but shows no signs of toxicity (Volles and Lansbury, 2007). H50Q, a fourth mutant, does not bind tightly to the membrane, which causes it to aggregate faster. More work is being done to see if mutation G51D has the same effect as H50Q (Ghosh et al., 2013). A sixth mutation, A53E, has been discovered recently in one family. The patient experienced early onset pathology, age 36, which is typical of familial PD (Pasanen et al., 2014). An abundance of SNCA, the gene that encodes for ɑ-synuclein, was found in the patient’s brain and spinal cord. This suggests A53E makes ɑ-synuclein aggregate aggressively (Pasanen et al., 2014). Researchers suggest A53E is the cause of the patient’s PD, however more research is needed to investigate A53E’s role in PD. Designing this gene will help us gain a better understanding into the role A53E plays in PD, as well as the effects C-terminus truncations have on ɑ-synuclein. It is known that the A53E mutation causes aggressive aggregation of the SNCA gene. Beyond this, the role of A53E in PD pathology is not yet understood. Also, the role of the C-terminus in ɑ-synuclein is still unclear. We hypothesize that the A53E mutation, along with the C-terminus truncation, will cause a more aggressive pathology than wild-type or fulllength ɑ-synuclein. We hypothesize that solubility will decrease and aggregation will increase, as more amino acids are being removed from the C-terminus of ɑ-synuclein when the protein is truncated. Thus, the 120 amino acid truncation will show more aggregation than the 123 amino acid truncation, as more amino acids will be removed from the 120 version compared to the 123 *This author wrote the paper as a part of BIOL221: Molecules, Genes, and Cells under the direction of Dr. DebBurman Primary Article Eukaryon, Vol. 11, March 2015, Lake Forest College version. In this study, yeast will be used to explore the role ɑ-synuclein plays in PD. Yeast is the first eukaryote for which the entire genome was mapped. This allows for easy manipulation of genes; genes can be taken out and added to yeast at will. As PD is a protein misfolding and degradation problem and yeast make, fold, and degrade proteins similar to humans, yeast are an excellent model for which to study PD (Allendoerfer et al., 2008). Yeast are also inexpensive, therefore an appropriate model for undergraduate research. Yeast have provided significant insights into PD, showing toxicity patterns for ɑ-synuclein as well as localization patterns of the protein inside a cell (Sharma et al., 2006; Fiske et al., 2011). When expressed in yeast, ɑ-synuclein has been shown to associate with the plasma membrane in a highly selective manner (Outeiro and Lindquist, 2003). In this study we aim to make two truncated versions of the familial mutant A53E of ɑ-synuclein (Figure 1C). We will make truncated versions 123 and 120 of familial mutant A53E, both of which will be tagged with green fluorescent protein (GFP) (Figure 1A). GFP will be used to show localization patterns inside the cell in future studies, in order to analyze the membrane binding and aggregation patterns of ɑ-synuclein compared to full-length and wild-type versions of the protein (Sharma et al., 2006).