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Parkinson’s disease (PD) is an incurable neuro- degenerative disease characterized by the death of midbrain dopaminergic neurons. The suspected cause of PD is the misfolding and aggregation of a brain protein, α-synuclein. A popular hypothesis is that increasing degradation of α-synuclein may protect the cell from aggregation and toxicity. While strong pharmacological evidence indicates that autophagy is involved in the degradation of α-synuclein in PD, the genetic link remains weak. For my senior thesis, I evaluated the hypothesis that the lysosome is the route for α-synuclein degradation by the autophagy pathway. Specifically, I studied the genetic link between autophagy and α-synuclein using a budding yeast model. My results demonstrate four significant findings. First, through genetic evidence, my results support the hypothesis that α-synuclein is regulated by the autophagy pathway. Of the six autophagy genes that I evaluated, deletion of each altered one or more α-synuclein PD-related properties. Second, these deletion effects on α-synuclein were gene-specific, indicating substrate-specificity. Third, α-synuclein localization exhibited a range of cellular distributions depending on the deleted gene. Finally, I did not observe α-synuclein dependent toxicity. Our lab’s cumulative data further supports the hypothesis that the lysosome is the route for α-synuclein degradation by the autophagy pathway.

Introduction

Autophagy

Self-cannibalism elicits alarming and horrifying images. Yet, believe it or not, self-cannibalism is a normal process that our bodies use daily, such as between meal times. Autophagy is the cell biologist’s term for such “self-eating.” This degradation mechanism is an evolutionarily conserved degradation process designed to help cells and organisms survive conditions of stress, starvation, and infection or illness (Shintani & Klionsky, 2004). Its primary role is to break down old or damaged proteins, protein complexes, and organelles, both to protect cells from toxicity and as a way to obtain energy by recycling cellular components.

The scientific field has come a long way to understanding the molecular mechanisms of autophagy. New research suggests that a balance in autophagy is key to systematic health and an imbalance can result in diverse human diseases (Fig 1A). These diseases include diabetes, Crohn’s disease, heart disease, aging, cancer and neurodegeneration.  For example, Crohn’s disease, an inflammatory disease of the gastrointestinal tract, is due to deficient autophagy. When autophagy is deficient, the organism responds by over-activating the inflammatory response leading to the symptoms of the disease (Cadwell et al., 2008).  On the contrary, too much autophagy can also result in diseases such as Pompe disease, where the outcome is the disruption of the contractile apparatus of the muscle fibers (Shea & Raben, 2009). This disease is made more difficult to treat because autophagy also interferes with the enzyme replacement therapy used to treat the disease (Shea & Raben, 2009). Overall, the wide range of diseases where autophagy plays a role demonstrates how integral this process is to the normal function of the cell. Thus, a balanced autophagy is critical for maintaining a healthy organism. My thesis explores the link between neurodegenerative diseases and autophagy.

Autophagy and Neurodegenerative Diseases

While autophagy is involved with many diseases, the research towards uncovering the role of autophagy in neurodegenerative diseases is still in its infancy. Neurodegenerative diseases are a class of disorders characterized by selective death of neurons in the central nervous system (Ross & Poirier, 2004). This death is believed to be due to the misfolding and aggregation of a protein particular to each disease (Fig 1B).  The death of neurons leads to the symptoms seen in each of the diseases such as motor deficits or memory loss. Research shows that mice deficient for an important autophagy gene, Atg5, develop abnormal intracellular protein accumulations, which then develop into aggregates and inclusions (Hara et al., 2006). This suggests that autophagy is necessary for continued clearance of cytosolic proteins that otherwise have the potential of accumulating and interfering with proper neuronal function that can lead to neurodegeneration.  Additional findings in mice show that the loss of Atg7, another essential autophagy gene, leads to behavioral defects, massive neuronal loss in the cerebral and cerebellar cortices, and accumulation of polyubiquitinated proteins (Komatsu et al., 2006). Furthermore, Atg7 is vital for proper membrane trafficking and turnover in the axons (Komatsu et al., 2007).  When Atg7 is deleted in the cerebellar Purkinje cells, axonal dystrophy and degeneration occur (Komatsu et al., 2007). Thus, this data suggests the importance of autophagy in axonal homeostasis.

Research demonstrates that suppression of autophagy in neuronal cells leads to neurodegenerative diseases (Komatsu et al., 2007). Conversely, induction of autophagy protects cells from toxicity in such diseases as Huntington’s (Ravikumar et al., 2004). For instance, Ravikumar et al. (2004) showed that induction of autophagy leads to protection against toxicity in both fly and mouse models. This neurodegenerative disease is characterized by a misfolding of a protein, huntingtin (Ross & Poirier, 2004). Similarly, in Parkinson’s disease, the culprit of the disease is a misfolded protein. Thus, what is the connection between autophagy and Parkinson’s disease?

Parkinson’s Disease

Parkinson’s disease (PD) is the second most common incurable progressive neurodegenerative disease after Alzheimer’s, affecting one million people in the United States and five million worldwide (Olanow et al., 2009). Unfortunately, the number of affected individuals is expected to double by 2030 (Olanow et al., 2009). The consequences of this will be a greater burden on the health care system and will increase the need for effective treatments (Dorsey et al., 2007).  PD can develop at any age, though it is most common in older adults, with the peak of onset at age sixty (Fahn, 2008). With increased age, the prevalence and

PD can be characterized by both motor and non-motor deficits (Fig 2A). Motor deficits include tremor-at-rest, bradykinesia, rigidity, loss of postural reflexes, flexed posture, and the freezing phenomenon (Fahn, 2008). Non-motor functions affected include cardiovascular and gastrointestinal abnormalities, cognitive dysfunction, and depression (Fahn, 2008).  Patients with PD usually live for twenty or more years post diagnosis and death is due to concurrent unrelated illness (Fahn, 2008).

Two forms of PD exist: sporadic and familial.  The most common form of PD is sporadic, which accounts for 85-90% of PD cases (Fahn, 2008). Current research on sporadic PD demonstrates the links between PD and oxidative stress, mitochondrial and proteasomal dysfunction, and environmental toxins (Fahn, 2008). Familial PD accounts for the remaining 10-15% of all cases (Fahn, 2008). To date, the field has identified the following mutations in these seven genes: Parkin (Matsumine et al., 1997), UCH-L1 (Leroy et al., 1998), PINK1 (Valente et al., 2001), DJ-1 (Bonifati et al., 2002), LRRK2 (Funayama et al., 2002), ATP13A2 (Najim al-Din et al., 1994; Hampshire et al., 2001), and α-synuclein (Polymeropoulos et al., 1996). Out of

all these familial genes, α-synuclein has been studied the most. The α-synuclein gene codes for a protein called α-synuclein, which has been linked to PD. The first α-synuclein

missense mutation identified was of alanine to threonine at amino acid 53 (A53T) in an Italian family (Polymeropoulos et al., 1997). The second mutation discovered was A30P (alanine to proline on amino acid 30) (Kruger et al., 1998). Finally, the most recent mutation is E46K (glutamic acid to lysine on the amino acid 46) (Zarranz et al., 2004).  This small change in the primary sequence of the α-synuclein has

a crucial effect, resulting in an α-synuclein mutant protein that has a higher rate of misfolding when compared to normal α-synuclein (Polymeropoulos et al., 1997; George, 2002). If α-synuclein misfolds, it can no longer sustain its function in a cell, which can ultimately lead to the death of the cell.

The Function and Properties of α-Synuclein

α-Synuclein is a 140 amino-acid cytosolic protein that was initially identified as a precursor protein for the non-β amyloid component of the amyloid plaques of Alzheimer’s disease (Ueda et al., 1993). The DNA sequence of α-synuclein is homologous to two other proteins: β-synuclein and γ-synuclein (Giasson et al., 2001). In vitro studies indicate that when either mutated α-synuclein or wild-type (WT) α-synuclein are expressed, aggregates of insoluble protofibrils form (Conway et al., 1998; Spillantini et al., 1998; El-Agnaf et al., 1998; Giasson et al., 1999), which are composed of β-sheets and amyloid-like filaments (El-Agnaf et al., 1998; Hashimoto et al., 1999; Narhi et al., 1999). Furthermore, mutated α-synuclein (A 53T or A30P) has a faster rat fibrilization at higher concentrations than WT α-synuclein (Conway et al., 1998), with the A53T mutant being the fastest. 

An insight into monomeric α-synuclein demonstrates that it does not contain any stable structure and has become known as a “natively unfolded” protein (Uversky, 2003). This implies that, due to its inherent plasticity, monomeric α-synuclein can adopt a series of conformations depending on its environment. However, in cases of increased concentration, α-synuclein undergoes self-assembly into dimers and small oligomers, which allows for its stabilization (Uversky, 2003). Interestingly, in Lewy bodies, α-synuclein is found in the form of protofibrils and small oligomers (Spillantini et al., 1997). This suggests that the formation of protofibrils is linked to cell death (Goldberg & Lansbury, 2000; Kaplan et al., 2003; Volles & Lansbury, 2003). Furthermore, both in vitro studies and ¬in vivo studies show that α-synuclein is found aggregated in neurons (Baba et al., 1998; Trojanowski et al., 1998). This aggregation, through an unknown mechanism, ultimately leads to neurotoxicity (Ding et al., 2002; Sharon et al., 2003; Periquet et al., 2007; Fig 2B). Moreover, α-synuclein is found highly modified through phosphorylation and nitration, and these modifications have been linked to toxicity (Shimura et al., 2001; Hodara et al., 2004; Fujiwara et al., 2002; Okochi et al., 2000). My colleague, Keith Solvang ‘11, investigated the properties of modified α-synuclein in his senior thesis.

Figure 2: Parkinson’s disease pathway.   A. In Parkinson’s disease, dopaminergic neurons die in the substantia nigra, which is part of the basal ganglia circuitry. The disruption of the basal ganglia, the part of the brain responsible for voluntary movement, leads to decreased excitation of the motor cortex neurons. This leads to the symptoms seen in PD such as resting tremor and difficulty initiating movement. B. Misfolded and aggregated α-synuclein is found inside these dying neurons and is thought to lead to cellular toxicity and ultimately cell death. While the reasons for misfolding and aggregation are still unknown, impaired degradation, over the years, has become one of the studied candidates.  

Parkinson’s disease is a gain-of-function disease, meaning that in PD, α-synuclein gains novel properties. When WT α-synuclein or mutant α-synuclein (A53T or A30P) is overexpressed, it is toxic to neuroblastoma cells and results in cell death (Ostrerova et al., 1999). Moreover, overexpression of WT α-synuclein in some transgenic mouse lines results in PD-like pathogenesis: development of cytoplasmic α-synuclein inclusions, loss of dopaminergic synapses, and motor impairment (Masliah et al., 2000). Furthermore, overexpression of A53T mutant α-synuclein in mouse lines results in severe motor defects that lead to paralysis, death, and appearance of insoluble α-synuclein and Lewy body-like inclusions (Giasson et al., 2002). In Drosophila, expression of human wild-type α-synuclein or mutant α-synuclein results in the formation of Lewy body-like inclusions located in the dopaminergic neurons, neuronal loss, and impaired climbing abilities, indicating difficulty in movement (Feany & Bender, 2000). Similar results have been seen in Caenorhabditis elegans (Lakso et al., 2003). Additionally, accumulation of α-synuclein is seen in the brain of PD patients (Xu et al., 2002; Devi et al., 2008; Shehadeh et al., 2009).

Another important property of α-synuclein is that it binds the plasma membrane and endomembrane within the cell. In yeast studies, WT and E46K α-synuclein is found to localize to the plasma membrane (Outeiro & Lindquist, 2003; Dixon et al., 2005; Sharma et al., 2006; Cooper et al., 2006).  Additionally, studies have demonstrated that α-synuclein binds to the inner membrane of mitochondria (Devi et al., 2008) and to the plasma membrane of vesicles (Kamp & Beyer, 2006). Lastly, studies demonstrate that overexpression of α-synuclein leads to mitochondrial dysfunction (Martin et al., 2006; Stichel et al., 2007; Su et al., 2010). In C. elegans, α-synuclein binds to mitochondria through an unknown mechanism, resulting in mitochondrial fragmentation. In this study, mitochondrial fragmentation could be rescued by coexpression of Parkin, DJ-1, or Pink1, each of which are the genes that, when mutated, are associated with familial PD (Kamp et al., 2010).  

Out of these three genes, Parkin codes for an E3 ubiquitin ligase involved in proteasomal degradation. Therefore, mitochondrial dysfunction, due to α-synuclein, can be rescued by proteasomal degradation.This suggests that studying α-synuclein degradation could uncover possibilities for cell survival.  α-Synuclein degradation is the primary focus of my thesis.  

Degradation of α-Synuclein

Proteins are one of the building blocks of life; they are able to perform diverse cellular functions that are driven by their specific amino acid sequences and resultant structures. Like all “machinery,” protein molecules can acquire wear and tear and become damaged as a natural consequence of cellular stress and age (Rubinsztein, 2006). To handle recycling of such damaged proteins, cells have evolved two degradation machines: the proteasome and the lysosome (Rubinsztein, 2006). The proteasome degrades short-lived proteins that are found in the nucleus or cytoplasm, while the lysosome

Figure 3:  The routes to the lysosome and the proteasome.  The figure demonstrates the three routes that lead to lysosomal degradation: phagocytosis, endocytosis, and autophagy. Phagocytosis degrades bacteria and viruses by engulfing them. In endocytosis, material from outside of the cell or from the plasma membrane is internalized into a vesicle and tagged to the lysosome for degradation. Finally, in autophagy, material that is found in the cytoplasm is internalized into an autophagosome that is transported to the lysosome for degradation. Additionally, the proteasomal degradation pathway is illustrated. With the help of El, E2, and E3 n d  enzymes, the material is tagged with ubiquitin and is recognized by the proteasome for degradation.

degrades proteins that are found bound to the plasma membrane. The question remains how α-synuclein is degraded. The localization of α-synuclein creates a challenge for its degradation because it is found in the Figure  cytoplasm, bound to the plasma membrane, and bound to vesicles (Kahle et al., 2000; Davidson et al., 1998; Eliezer et al., 2001). Recent studies have shown that both WT and A53T are delivered to the cell’s periphery via the secretory pathway, while A30P does not enter the secretory pathway and is found diffuse in the cytoplasm (Dixon et al., 2005). Furthermore, α-synuclein is found in body fluids, which means that some amount is secreted outside the cell (Lee et al., 2008).  Thus, since α-synuclein localizes to different parts of the cell, both the proteasome and the lysosome are likely involved in its degradation (Fig 3). I will first focus on the proteasome and then on the lysosome.

Proteasomal Degradation          

The proteasome is a large, barrel-shaped catalytic complex that is part of the ubiquitin-proteasome system (UPS; Fig 3). This mechanism degrades intracellular soluble proteins that are found in the cytoplasm, nucleus, and endoplasmic reticulum and are mutated, misfolded, denatured, misplaced, or damaged (Davies, 2001; Goldberg, 2003). Over the years, compelling evidence suggests that UPS dysfunction is linked to Parkinson’s disease. In familial PD, patients with either of two mutated genes, Parkin (Matsumine et al.. 1997) or UCH-L1 (Leroy et al., 1998), exhibit PD-like symptoms, and postmortem analyses demonstrate formation of Lewy bodies. In sporadic PD, proteasome structure and function is altered in the substantia nigra (McNaught et al., 2003). In vitro studies demonstrate that inhibition of the proteasome results in a selective degeneration of dopamine neurons paired with formation of inclusions that are stained positively for both α-synuclein and ubiquitin (McNaught et al., 2002;   Rideout et al., 2005). Furthermore, injections into the substantia nigra or striatum of proteasomal inhibitors induce dopamine neuron BBB.  

degeneration with inclusions in rats (McNaught et al., 2002; Fornai et al., 2003). In rats, systematic administration of proteasome inhibitors leads to the development of PD-like symptoms with PET scans demonstrating progressive loss of dopaminergic nerve terminals in the striatum. Postmortem analyses of these animals display striatal dopamine depletion and formation of inclusions in the substantia nigra (McNaught et al., 2004).  

Numerous studies have demonstrated that α-synuclein is degraded by the proteasome (Bennett et al., 1999; Tofaris et al., 2001). Specifically, wild-type α-synuclein has been shown to be a substrate for both the 20S and 26S proteasomes and degraded mostly in an ubiquitin-independent manner. However, the story differs for the A53T α-synuclein mutant. Unlike WT, both in vivo and in vitro studies established that the A53T α-synuclein mutant protein does not undergo proteasomal degradation (Stefanis et al., 2001; Tanaka et al., 2001). These studies serve as compelling evidence that α-synuclein is degraded by the proteasome, but also suggest the occurrence of other types of α-synuclein degradation, particularly for the mutant protein.

Lysosomal Degradation

The journey of α-synuclein degradation does not end with the proteasome. In 2003, Webb et al. hinted that the lysosome is also involved. Three pathways to the lysosome exist: phagocytosis, endocytosis, and autophagy (Fig 3). Viruses and bacteria use the phagocytosis pathway; this pathway is not used by α-synuclein. Substantial evidence in the field suggests that endocytosis is the route for α-synuclein degradation (Willingham et al., 2003; Kuwahara et al., 2008; Perez Thesis, 2010; Ayala Thesis, 2009). However, autophagy also appears to have a role. When Webb et al. (2003) specifically inhibited autophagy, α-synuclein accumulated in the cell. Chemical induction of autophagy with rapamycin leads to decreased α-synuclein levels, as compared to the control. Three types of autophagy exist: microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. In microautophagy, the whole organelle or portion of the cytosol is sequestered directly by the lysosome for degradation. CMA involves the direct translocation of cytosolic proteins that contain a particular pentapeptide motif to the lysosome (Rubinsztein, 2006). Substantial evidence in the field exists that CMA is one of the pathways used by α-synuclein (Cuervo et al., 2004; Vogiatzi et al., 2008). When the lamp2a gene, which codes for a lysomal receptor for CMA, is deleted, α-synuclein accumulates (Vogiatizi et al., 2008). Macroautophagy degrades the bulk of cytoplasmic proteins and organelles found in the cytoplasm (Rubinsztein et al., 2005; Fig 4). When macroautophagy is pharmacologically inhibited, α-synuclein accumulates (Vogiatzi et al., 2008), which suggests that this mechanism (hereafter referred to as autophagy) also degrades α-synuclein. Furthermore, expression of mutant α-synuclein in cells results in the accumulation of autophagic-vesicular structures, suggesting that autophagy is involved in the degradation of mutant α-synuclein (Anglade et al., 1997; Stefanis et al., 2001).  Moreover, Prigione et al. (2010) demonstrated an induced autophagy response in peripheral blood mononuclear cells of PD patients while α-synuclein in these cells was in a soluble form. They showed that the lack of aggregated α-synuclein can not be due to the UPS activity, since increased activation of the system was not detected. Thus, these cumulative studies further support the hypothesis that the autophagy route degrades α-synuclein. 

Figure 4: Autophagy pathway.  The figure demonstrates the three steps of the autophagy pathway: nucleation, expansion, and fusion. In the nucleation step, the membrane begins to form around the material that needs to be degraded. In the expansion step, the autophagosome forms. In the fusion step, the autophagosome fuses with the lysosome. In each of the steps, there are numerous proteins involved that are organized into complexes. Complex I and II operate in the nucleation step. Complex I regulates induction of autophagy, followed by vesicle nucleation (as demonstrated by the red arrow) regulated by complex II. Complexes III, IV, and V operate in the expansion step. Complex II and complex III work together to expand the vesicle by adding more plasma membrane. Complex V retrieves Atg9 from the plasma membrane, preparing the autophagosome for the fusion step. The fusion step is regulated by a different set of proteins.  For my thesis, I studied genes from complexes I, II, III, IV, and V. The figure is adapted from Nakatowa et al. (2009).

Autophagy proceeds in three steps: nucleation, expansion, and fusion with the lysosome (Fig 4). The nucleation step is composed of the membrane starting to form around the items that need to be degraded. During expansion, an autophagosome forms. Finally, during the fusion step, the autophagosome fuses with the lysosome for the degradation to occur. The conventional method of studying autophagy has been through the discovery of autophagy genes (Shintani &  Klionsky, 2004; Suzuki & Ohsumi, 2007).  A new, elaborate way of thinking about autophagy genes is by grouping them into complexes (Fig 4). In particular, five main complexes are involved in the process. When autophagy is induced through either starvation or rapamycin, the following complexes form: Atg1 kinase complex (Complex I), Ptdlns 3-kinase complex (Complex II), Atg8 conjugated system complex (Complex III), Atg12 conjugated system complex (Complex IV), and Atg2-Atg18 complex and Atg9 (Complex V). Complex I and Complex II are involved with the initial step of nucleation. Once autophagy is activated, the proteins involved in Complex I bind to the lipid membrane and work with Complex II to attract other autophagy proteins to the preautophagosomal structure (PAS). The function of Complex III and Complex IV is to continue building the plasma membrane until it forms the autophagosome. Once the autophagosome is complete, Complex IV removes Atg9 from the membrane, and the autophagosome is ready for fusion with the lysosome.Figure 5: Predictions and genes evaluated.  A. I evaluated three properties of α-synuclein in autophagy deficient strains compared to the autophagy intact strains: toxicity, localization, and expression. 1) For the toxicity analysis, I predicted that I would see more cell death in the autophagy deficient strains since Iwill see fewer cells (black circles) on the plate as compared to when autophagy is intact. 2) For the localization analysis, I predicted that WT and E46K α-synuclein (represented by green) will change localization. In autophagy intact cells, WT and E46K α-synuclein localizes to the plasma membrane. In autophagy deficient cells, I predicted that WT and E46K α-synuclein would become cytoplasmically diffuse and form some aggregates.  A30P α-synuclein would stay cytoplasmically diffuse and form many aggregates. In WT, E46K, and A30P, I predicted the vacuole (represented by V) will stay dimly lit. 3) For expression analysis, I believed α-synuclein would accumulate in the autophagy deficient strains as compared to the autophagy intact strain (as demonstrated by a cartoon of a Western blot). B. The genes in red are the novel genes that I examined. The genes in black are those that have already been evaluated by our lab but were re-assessed by me once again. The genes in blue were not studied.

For my thesis, I evaluated complexes I, II, III, IV, and V. In particular, I evaluated Atg29 and Atg31 in complex I, Atg6 and Atg14 in complex II, Atg7 in complex III (which is also found in complex IV), and Atg9 in complex V. Atg29 and Atg31 cooperate with Atg17 for the recruitment of other Atg proteins to PAS. Atg6, also known as Vps30, has an unknown function but is known to be one of the substrates of the complex. Atg14 is responsible for the recruitment of Ptdlns 3-kinase complex to the PAS and is required for localizing additional Atg proteins to the PAS. Atg7 belongs to the E1 family of enzymes, which are ubiquitein-activating enzymes. Finally, Atg9 is a transmembrane protein that is involved in autophagic vesicle formation. While the exact function is unknown, Atg9 may be involved in membrane delivery to the PAS.

Gap in Knowledge

While pharmacological and genetic evidence exists in support of proteasomal degradation of α-synuclein, only pharmacological evidence exists in support of autophagy. In order to demonstrate that autophagy is involved, further genetic evidence needs to be obtained. Evaluation of all of the genes involved in the pathway is vital to understanding autophagy’s role because this approach would demonstrate if some genes are more important than others in the guidance of α-synuclein through this degradation pathway. Furthermore, evaluation of all of the autophagy genes will demonstrate whether a substrate dictates the specificity of interactions with the proteins coded by these genes. Since my lab is interested in substrate selective therapy, determining which genes are critical for α-synuclein regulation is important. Moreover, genetic screens need to be performed, but they need to evaluate all three properties seen in PD pathology: toxicity, altered localization, and accumulation.  Ray Choi ‘09 began the first genetic study of autophagy genes in a budding yeast model (Table 1). My thesis targets the remaining autophagy genes to fill the gap of the lack of genetic evidence to support autophagy as a route of degradation of α-synuclein.           

Our Lab’s Model Organism

Numerous animal models are used to study the role of α-synuclein in PD. These models include flies (Feany & Bender, 2000), mice (Giasson et al., 2002), worms (Lakso et al., 2003), and yeast (Outeiro & Lindquist, 2003). Yeast is a single cell fungus that has a short generation time, fully sequenced genome, and well-established molecular genetics (Kaeberlein et al., 2001). Most of the pathways in yeast are evolutionarily conserved between higher eukaryotes such as humans (Kaeberlein et al., 2001). Autophagy was first characterized in yeast, and to date, 33 Atg genes have been identified in budding yeast that are responsible for the three steps of autophagy (Klionsky et al., 2003; Suzuki et al., 2007; Ventruti & Cuervo, 2007). Also, yeast is a relatively inexpensive model organism and easy to work with in an undergraduate setting. Our lab uses budding yeast as a model to study α-synuclein (Sharma et al., 2006; Brandis et al., 2006). α-Synuclein is not toxic to budding yeast cells, and wild-type α-synuclein localizes predominatly.

The summary chart summarizes all of the genes that have been evaluated thus far in our lab. The genes were originally evaluated by the following: Ray Choi ‘09: atg1∆, atg2∆, atg8∆, atg17∆, atg18∆, vps15∆; Danny Sanchez ‘11: atg3∆, atg4∆; Jaime Perez ‘10: vps34∆; Kayla Ahlstrand ‘12: vam3∆, vam4∆; Peter Sullivan ‘12: atg11∆, atg13∆

to the plasma membrane (Sharma et al., 2006). However, other labs have seen α-synuclein dependent toxicity in budding yeast (Outeiro & Lindquist, 2003).  We use single gene deletion strains to study α-synuclein-dependent toxicity, localization, and expression. Recently, research was conducted on autophagy by the DebBurman lab, and the researchers saw that when autophagy is compromised, α-synuclein is not toxic to the cells (Choi Thesis, 2009; Table 1). In some particular deletion strains, a change in localization and increased expression of α-synuclein is seen (Choi Thesis, 2009). To date, no studies have evaluated autophagy in budding yeast.