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In 1817, a British apothecary James Parkinson, published an essay: An Essay on the Shaking Palsy reporting 6 cases of paralysis agitans and described the symptoms and how the disease evolves over time, which awarded him by naming the disease bearing his name .
Attributed to the degeneration of dopaminergic (DA) neuronal cells in the substantia nigra pars compacta (SNpc), Parkinson's disease (PD) is a chronic age-related central nervous system disorder caused by progressive neurodegeneration [2-4].
Movement-related symptoms such as tremor, rigidity, slowness of voluntary movement, postural instability, akinesia, gait and balance problem, dysarthria and dysphagia appear as PD progresses over time . In those suffering from the disease for more than ten years, non-motor symptoms like cognitive decline, dementia and sleep-wake cycle dysregulation may also occur.
Although some atypical cases (10%) are ascribed to heritable genetic mutations, most cases of Parkinson's disease are still sporadic . The pathology of the disease is characterized by the selective death of dopaminergic neuronal cells in the SNpc, along with the abnormal intracellular proteinaceous aggregates called Lewy bodies (LBs) and Lewy neurite (LNs), which serve as hallmarks of Parkinson's disease.
So far there is no proof showing that either cell loss or the presence of Lewy bodies alone can be used for PD. The investigation of both of them, however, throws light upon the disease diagnosis. The distribution of Lewy bodies varies individually throughout the Parkinsonian brain, and is often directly linked with the degree of the PD symptom of each individual.
Treatment that significantly decelerates the progression of the disease is still under research. However, effective therapies, which could relieve the early motor symptoms of the disease with dopamine replacement strategies, such as levodopa (L-Dopa) and dopamine agonists (DA- agonists) were reported . With the progression of the disease and the continuing loss of dopaminergic, these drugs eventually become less efficient and an involuntary writhing movement complication, dyskinesia occurs.
Several studies have demonstrated that exposure to insecticides, pesticides and herbicides as rotenone and paraquat, which elicit neurodegenerative phenotypes, can contribute to PD [7-9]. Derived from the roots of various plants, Rotenone is an organic compound that is used to eradicate nuisance fish and insect population. Being transported into the brain very rapidly, rotenone is also a classical, well-characterized and high affinity specific inhibitor of mitochondrial NADH dehydrogenase (complex I), one of the key enzyme complexes of the inner mitochondrial membrane, where oxidative phosphorylation takes place [10, 11]. Independent from the dopamine transporter for access to the cytoplasm of dopaminergic neuron, Rotenone crosses biological membranes easily due to its hydrophobicity [12, 13]. Symptoms of Parkinsonism such as bradykinesia and rigidity appear from animals after Rotenone infusing. Flexed posture and typical paralysis of advanced PD could also be found from seriously affected rats, showing that chronic, systemic rotenone infusion recapitulated the anatomical, biochemical, pathological and behavioral are features of PD 
Despite the fact that Rotenone has been correlated to PD, treatment as a new method for selective nigrostriatal damage is still controversial. On the one hand, Rotenone has been shown to lead to selective dopaminergic cell death in vivo. Selective nigrostriatal dopaminergic degeneration was reported in rotenone infused intravenously or subcutaneously rat, although rotenone also induced degeneration of non-dopaminergic neurons in both the basal ganglia and the brainstem [12, 15, 16]. On the other hand, as for in vitro models, it was reported that dopaminergic neurons have higher sensitivity to rotenone-induced toxicity than other neuronal cells [17-19], but non-dopaminergic neurons were reduced by rotenone in primary mesencephalic cultures as well [20-22].
1.2 The Introduction of Î±-Synuclein
1.2.1 Parkinson's disease caused by Î±-Synuclein
The protein of Î±-synuclein, encoded by the Î±-synuclein gene (SNCA), is a small presynaptic phosphoprotein with 140 amino acids , extensively localized in the nucleus of mammalian brain neuron [3, 23]. Normally, as an unstructured soluble protein, Î±-synuclein is found to be misfolded which aggregates to form insoluble fibrils as a major component of Lewy bodies [3, 4, 24]. The dysfunction finally results in dopaminergic neuronal death and is associated with both sporadic and familial pathogenesis of several neurodegenerative diseases (e.g. Parkinson's disease, dementia and multiple system atrophy).
The missense mutations or overexpression of Î±-Synuclein (i.e. A53T, A30P, E46K and gene triplication) were reported to result in cytotoxicity, which causes direct cell loss and maybe dominant inherited PD [4, 23, 25]. A53T mutation was the first clearly reported Mendelian gene that can cause PD .
Althoug the phenomenon that Î±-synuclein is closely related to Parkinson's disease is widely reported, the mechanism of Î±-synuclein has not been clearly understood yet. However, a molecular study in 2008 suggests that Î±-synuclein exists as a mixture comprised of unstructured, Î±-helix and ï¢-sheet-rich conformers in equilibrium. The aggregation mechanism of Î±-synuclein is still under controversy .
1.2.2 Cytotoxicity of Î±-Synuclein
The expression of Î±-synuclein negatively impacts yeast cells (Saccharomyces cerevisiae) in growing and stationary phases. It has been reported to effect invertebrate model animals ï¼ˆe.g. Drosophila melanogaster and Caenorhabditis elegansï¼‰as well, when the introduction of high level production of mutant forms or increase production of Î±-synuclein damages dopaminergic neuron cells in vivo and in vitro. All three models showed negative impact on cells with the presence of Î±-synuclein in the organisms where it's normally not present. Additionallyï¼Œ cell trauma and death appeared as a result of high levels of Î±-synuclein or its mutant forms when produced in primary neurons of mammalian cell culture in vitro .
1.2.3 Reason for Cytotoxicity of Î±-Synuclein
Figure Events in Î±-synuclein toxicity 
The major pathway for protein aggregation can be illustrated by the left panel of Figure 1. As a natively unfolded structure, monomeric Î±-synuclein can bind to membranes in the form of Î±-helix, and the two forms ï¼ˆunfolded and Î±-helix ) transform to each other in equilibrium within the cell. The in vitro work further clarifies that the unfolded monomer tends to aggregate into small oligomeric sheets which can be stabilized by ï¢-sheet-like interactions at the first stage, and forms higher molecular weight insoluble fibrils later. In some neurons, pathological structures such as Lewy bodies may be formed as a result of the accumulation of Î±-synuclein. Both the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP) where mainly macroautophagy and chaperoninen mediated autophagy occur, have been suggested to contribute to Î±-synucleinÂ turnover.Â The presence of ubiquitin (shown as a black spot ) in Lewy bodies is believed to be part of a secondary process of accumulation, possibly due to increased unfolding of Î±-synucleinÂ which may enhance deposition in the growing Lewy body. The proposed cellular target for toxicity pertaining to Î±-synuclein, including ER-Golgi transportion, synaptic vesicles, mitochondria and lysosomes and other proteolytic machinery are clarified by the right panel of the figure above .
In Î±-synuclein transgenic models, Î±-synuclein aggregation is always related to neuronal degeneration either by the loss of function or by death . Acknowledged from previous work, Î±-synuclein can form small oligomers inside the living cells, which may be associated with cytotoxicity. The formation of Î±-synuclein oligomers is linked to cell membrane damage and results in the dysfunction of multiple cellular systems.
Since Î±-synuclein may also play an important role in mediating both mitochondrial function and damage, cells which are Î±-synuclein overexpressing tend to display considerable mitochondrial dysfunction, including increase of protein oxidation, increase of reactive oxygen species (ROS) production, loss of mitochondrial membrane potential and reduction of Complex I activity [27-29]. Research shows that Î±-synuclein localizes in the mitochondria inner membrane conditionally. The Î±-synuclein protein is highly expressed in the mitochondria in most parts of the brain where the cytosolic form is predominant. In addition, mitochondrial dysfunction is commonly observed with high levels of Î±-synuclein or its mutant forms, suggesting that Î±-synuclein has a negative impact on the biological function of mitochondria. Mitochondrial dysfunction is known to be a dominant contributor to PD due to the consistent observation of mitochondrial complex I ineffectiveness in PD patients. In this case, the background level of mitochondrial Î±-synuclein may be a potential factor affecting mitochondrial function and predisposing some neurons to degeneration. Î±-synuclein is not only known to interact with the lipid membrane directly, especially with the vesicles , but it has also been found that several genes which deal with metabolism of lipid play a role in Î±-synuclein toxicity by yeast genome screening. It has been reported that Î±-synuclein forms an extended helical structure on small unilamellar vesicles and is involved in vesicle trafficking by binding small vesicles. Indeed, there is growing evidence that Î±-synuclein is involved in normal protein trafficking at the Golgi and endoplasmic reticulum (ER) .
In the normal brain, immunostaining of Î±-synuclein shows a diffuse pattern of reactivity throughout the neuropil which is consistent with a predominantly synaptic localization. Immunostaining is used for the location study of Î±-synuclein. However, in PD brains, Î±-synuclein antibodies display extremely high sensitivity for LB and LN staining. To show this high sensitivity, Î±-synuclein staining is used more widely instead of the less specific staining by the dye eosin. Although it has been proved that Î±-synuclein has a close relation with PD, it is also reported that increasing production of some specific biological molecules, like heat shock proteins, (Hsps) can prevent toxic effects of Î±-synuclein in yeast and in flies [32, 33].
1.3 The Introduction of Yeast
The yeast model is probably the simplest system used to illustrate the toxicity caused by Î±-synuclein expression. With the best-characterized eukaryotic genome, budding yeast (Saccharomyces cerevisiae) is regarded as the ideal species for rapid genetic interrogation . Cell-autonomous mechanisms of neurodegeneration were also examined by using yeast as an interactome system, not only due to its simplicity and fully-understood genome, but also because it is easy to analyze an eukaryotic organism with the availability of genetic banks of diploid and haploid gene deletion screens . As a powerful genetic tool, budding yeast has been adopted to reveal protein misfolding processes underlying human diseases , including Huntington's disease [35, 36], prion disease , and amyotrophic lateral sclerosis [38, 39]. Full understanding of these biological systems and processes in human cells and mammalian model animals obviously has been helped by the understanding of that which occurred in these yeast cells.
Previous publications have already shown that the increasing Î±-synuclein expression could contribute to cell death in growing and stationary phase cultures . As yeast is unicellular with a scalable system, it enables high throughput genetic and small-molecule screens. Apart from the advantages we mentioned above, the most important advantage is the genetic tractability which enables its DNA to be transformed quite easily, while at the same time the homologous recombination is efficient [39, 40]. This facilitates both the replacement of any gene with a mutant allele and the recovery of almost any mutation created in vivo . These gene deletion "knock outs" of central biological processes (in haploid strains), such as DNA replication, are unfortunately lethal, but "conditional" or impaired systems may sometimes be constructed to partially examine some of the central or core processes.
It is nevertheless surprising that budding yeast is able to productively imitate some complex disease processes, such as those contributing to neurodegeneration. Thus yeast plays a non-substitutable role in initially understanding aspects of various neurodegenerative diseases. Major aspects of cellular pathology are captured by yeast models as the first step, and are providing novel gene-environment connections and therapeutic targets on an unprecedented scale through the use of high-throughput genetic based screens . Two characteristics are required for the model systems to be applied to human disease research: the model has to be relevant to human disease and the model has to be easily analyzed. Both of these characteristics are possessed by yeast, making it the best of choice in our research. The relevance can be ascribed to its conserved genome and cellular biology, while the convenience for analysis can be related to its genetic tractability, scalability and shorter generation times .
The amenability to analysis can be explained by its highly reproductibility, genetical stability, short generation time and easily transformed genome allowing either the addition or deletion of genes through homologous recombination, [41, 42]. Genomic homology is a phenomenon reported by several articles. At least 60% of yeast genes show robust human homologues or at least one conserved domain with human genes statistically [41, 42]. In addition, a close yeast homologue can be found among more than 25% of known human disease genes [43, 44], which is explained partially by the conservation of many fundamental cell biological processes between yeast and mammalian cells .
Besides genome, the cellular protein additionally demonstrates high conservation of sequence and functional applications, and more remotely, their interactive partners. The first clue to the function of many higher eukaryotic genes can be provided by the homology to a yeast gene. Numerous cellular pathways of high relevance to neurodegeneration, such as protein folding, quality control and degradation, vesicular trafficking and fusion, apoptosis, mitochondria and oxidative stress and autophagy, are conserved in yeast. The most common neurodegenerative diseases, (e.g. Alzheimer's disease and Parkinson's disease) are ascribed to intracellular proteinacious aggregates. Protein misfolding, oligomerization and aggregation with neurodegeneration have been tightly associated with multiple lines of evidence. Thanks to the conservation of the cellular protein quality system, these processes are readily studied in the yeast genetic systems .
In yeast, as in mammalian cells, the central organelle for the production of reactive oxygen species (ROS), which is heavily implicated in neurodegeneration, is the mitochondria as mentioned previously. The fermentative growth ability of yeast allows the analysis of mitochondrial defects that would be lethal in mammalian cells .
By adopting the yeast model, a conserved link between endoplasmic reticulum (ER)-Golgi traffic and Î±-synuclein toxicity has been established [34, 45]. The pathway proteins are translocated from the endoplasmic reticulum (ER) to the Golgi complex and then transferred by vesicles to the plasma membrane. This is of particular importance for neurons that need to transport proteins over long distances to nerve terminals and that release neurotransmitters by vesicular fusion . Additionally, yeast is increasingly recognized as a model organism for the study of apoptosis, as the basic molecular machinery executing cell death is phylogenetically conserved. In addition, yeast apoptotic death occurs in the dependence of complex apoptotic scenarios such as mitochondrial fragmentation, Cytochrome C release and cytoskeletal perturbations . The conserved mechanisms of yeast cell death and survival are relevant to neural loss. Both apoptotic and non-apoptotic cell death mechanisms are implicated in neurodegeneration .
The genetic tractability and genomic stability endow the yeast with significant advantages over transformed or cancer derived immortal mammalian cell culture lines, and these characteristics are particularly necessary in a model system for initial screening for therapeutic treatment of neurodegenerative diseases. Yeast phenotypes can be screened based on genome-wide deletion, reduced expression or overexpression libraries. Depending on whether yeast homologue exists or not, one of two general approaches can be employed to imitate human disease in yeast. The human disease-related gene can be disrupted or overexpressed to determine the loss or gain of function phenotypes respectively with the presence of homologue in yeast [39, 40]. It is also clear that yeast homologues exist in many genes associated with neurodegeneration. This modeling approach has already been proved to be highly productive .
1.4 The Introduction of High-throughput screening
The introduction of high-throughput scrrening originated from natural product screening 30 years ago, just for pharmaceutical, biological, biotechnological research and development. High-Throughput Screening (HTS) is a scientific method of experimentation particularly used in the process of drug discovery nowadays. The goal is to obtain a high efficiency by conducting copious parallel biological experiments or tests using automation and miniaturization techniques, with the use of robotics, liquid handling devices, sensitive detectors, and data processing and control software [47-49]. A great many experiments of genetic, pharmacological, and chemical assays in a much shorter duration can be realized by High-Throughput Screening, through which active compounds, antibodies or genes which modulate a particular biomolecular pathway can be rapidly identified, and the role of the interaction of a particular biochemical process in biology can be understood.
High-throughput screening is a key link in the chain comprising the industrialized drug discovery paradigm. In this procedure, as long as a compound interacts with a target in a productive way, it then passes the first obstacle to become a drug. Compounds that fail the primary screen go back into the library for further screening against other targets.
Compounds with desired results in a high-throughput screening, usually called hits, are collected for further testing to go through effect determination.
1.5 Blueberry and Parkinson's Disease
Numerous studies suggested that a decreased antioxidant/oxidative/inflammatory stress balance plays the most important role in mediating the deleterious effects of aging on behavior and neuronal function . From epidemiological evidence, it is indicated that antioxidant supplementation provides neuroprotection against age-related neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease . Experimental evidence obtained from animal model studies links consumption of specific fruits and vegetables to reduced age- and disease-related neurodegeneration. Higher levels of antioxidant activity may be involved in the putative protective effects. Blueberries (BBs) contain the foods highest levels of polyphenolic flavonoids .
A 20-year investigation revealed that greater intakes of berries were strongly associated with a lower risk of PD and blueberries were proved to be neuroprotective in primary midbrain cultures. Administration of berry fruits, which contain flavonoid, revealed protective effects on dopamine neurons from oxidative damage and apoptosis and inhibits the formation of Î±-synuclein fibrils , which clearly clarified that anthocyanins have a protective effect on PD in terms of epidemiology.
A large quantity of studies in the past 10 years have revealed that polyphenols can prevent or reduce the deleterious effects of oxygen derived free radicals associated with several chronic and stress related human and animal diseases, both in vitro and in vivo. Flavonoids are the largest group of polyphenols, which is mainly divided into anthocyanins, glycosylated derivative of anthocyanidin .
CHAPTER II MATERIALS AND METHODS
2.1 Yeast Culturing
2.1.1 Yeast Strains
Three Î±-synuclein overexpressing yeast strains from Dr. Susan Lindquist (Whitehead Institute for Biomedical Research, Howard Hughes Medical Institute), were studied in the experiment: Yeast strain W303 is the parent strain that served as a control in this project, while yeast strain cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) with single copy of Î±-synuclein gene and W303-CFP+YFP strain with two copies of Î±-synuclein gene were adopted as experimental groups .
The strains were constructed by integrating vectors pRS306 Ura marker and pRS304 Trp marker into the corresponding loci of W303 genome. (e.g. pRS306 was integrated into the Ura3 locus of W303). Uracil (Ura) is a nucleic acid and Tryptophan (Trp) is an amino acid, both of which are necessary for the growth of yeast strains in which the TRP and URA genes have been insertionally inactived. The integrated W303 strain requires the supplementation of these components into the medium, or both of these genes must be transformed into the strain to eliminate the need for these nutrients. The genotypes of the four strains used are listed below.
Table Genotypes of yeast strains W303, W303-CFP, W303-YFP and W303-CFP+YFP