Parkinson's disease (PD) is a common neurodegenerative disorder disease all over the world that mostly affects individuals over the age of 50. It is caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Overexpression and mutation of Î±-Synuclein have been identified as direct cause of cell loss and maybe dominant inherited PD, although the mechanism is not clear yet. Multiple biological models were used in the research to elucidate the mechanisms and to develop new therapies of this disease. In the present study, we screened an established yeast model induced by galactose and marked with a fluorescent protein for the Î±-Synuclein protein overexpression-induced toxicity, which can cause yeast cell death. Blueberries were previously shown to be neuroprotective in primary midbrain cultures. To find out which part in blueberry had protectively in neuro cells, the blueberry compound library was screened by the high-throughput screening to determine effective protective compounds to rescue the cell loss caused by the Î±-Synuclein protein. The hits in the yeast screen will be applied to protecting the SHSY5Y cells treated with rotenone which is one research model for parkinsonism disease in the future.
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Key words: Parkinson's disease, Î±-Synuclein toxicity, yeast, high-throughput screen, cell death rescue
CHAPTER I INTRODUCTION
1.1 The Introduction of Parkinson's disease
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) neurons in the substantia nigra pars compacta (SNpc), Parkinson's disease (PD) is a chronic age-related central nervous system disorder due to progressive neurodegeneration .
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 . 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 neurons in the substantia nigra pars compacta, along with the abnormal intracellular proteinaceous aggregates called Lewy bodies (LBs) and Lewy neurite (LNs) which serve as hallmarks of ParkinsonHYPERLINK "http://en.wikipedia.org/wiki/Parkinson's_disease"'HYPERLINK "http://en.wikipedia.org/wiki/Parkinson's_disease"s disease. PD brain predisposes even young cells to form Lewy bodies.
Neither cell loss nor Lewy bodies are respectively sufficient for PD diagnosis, the investigation of both of them, however, throws light upon the disease diagnosis. The distribution of the Lewy bodies throughout the Parkinsonian brain varies individually which is often directly linked with the expression and degree of the clinical symptoms of each individual.
Treatment that significantly decelerates the progression of the disease is still under research. Effective therapies, however, could relieve the early motor symptoms of the disease with dopamine replacement strategies, such as levodopa (L-Dopa) and dopamine agonists (DA- agonists) . With the progressing of the disease and the continuing loss of dopaminergic, these drugs eventually lose efficiency at treating the symptoms and at the same time an involuntary writhing movement complication, dyskinesia occurs.
Several studies have demonstrated that insecticides, pesticides and herbicides exposure such as rotenone and paraquat, which elicit neurodegenerative phenotypes can contribute to PD . Derived from the roots of various plants, Rotenone is an organic compound that used to eradicate nuisance fish and insects 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 enzymes complexes of the inner mitochondrial membrane oxidative phosphorylation takes place . Independent from the dopamine transporter for access to the cytoplasm of dopaminergic neuron, Rotenone crosses biological membranes easily due to its hydrophobicity . Symptoms of parkinsonism such as bradykinesia and rigidity appeares from animals after Rotenone infusing. Flexed posture and typical paralysis of advanced PD could also be found from seriously affected rats, showing chronic, systemic rotenone infusion recapitulated the anatomical, biochemical, pathological and behavioral features of PD
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Despite the fact that Rotenone has been linked with PD, it treated 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 . 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 , but non-dopaminergic neurons were reduced by rotenone in primary mesencephalic culture as well .
1.2 The Introduction of Î±-Synuclein
1.2.1 Parkinson's disease caused by Î±-Synuclein
The protein Î±-synuclein, encoded by the Î±-synuclein gene (SNCA), is a small presynaptic phosphoprotein with 140 amino acids , extensively localizes in the nucleus of mammalian brain neuron . As an unstructured soluble protein normally, Î±-synuclein was found to be misfolded which aggregates to form insoluble fibrils as a major component of Lewy bodies . The dysfunction finally results in dopaminergic neuronal death and associated with both sporadic and familial pathogenesis of several neurodegenerative diseases, such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy.
The missense mutations or overexpression in 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 . A53T mutation was the first clear report that a Mendelian gene could be a cause of PD .
The phenomenon that Î±-synuclein is closely related to Parkinson's disease is widely reported, while the mechanism of Î±-synuclein has not been clearly understood yet. Although a molecular study in 2008 suggests Î±-synuclein exists as a mix of unstructured, alpha-helix, and beta-sheet-rich conformers in equilibrium, there is considerable uncertainty on the aggregation mechanism of Î±-synuclein remains .
1.2.2 Cytotoxicity of Î±-Synuclein
The expression of Î±-synuclein negatively impacts yeast cells (Saccharomyces cerevisiae) in growing and stationary phases. It also has been shown to effect invertebrate model animals as well, Drosophila melanogaster and Caenorhabditis elegans, 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 have also been reported. All the three model systems showed negative impact on cells with presence of Î±-synuclein in the organisms where it's normally not present. Additionally cell trauma and death appears with the presence 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 could be illustrated by the left panel of the figure show above. As a natively unfolded structure, monomeric Î±-synuclein can bind to membranes in form of Î±-helix, and these two forms transform with each other in equilibrium within the cell. The in vitro work further clarifies that unfolded monomer tends to aggregate into small oligomeric sheets which can be stabilized by b-sheet-like interactions first followed by forming higher molecular weight insoluble fibrils. The accumulation of Î±-synuclein into pathological structures such as Lewy bodies is probably a late event that occurs in some neurons. Both theÂ ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP) where mainly macroautophagy and chaperoninen mediated autophagy 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 transport, synaptic vesicles, mitochondria and lysosomes and other proteolytic machinery are clarified by the right panel of the figure above .
Although it is widely acknowledged that Î±-synuclein is a cytoplasmic protein that exerts its pathogenic effects in the cytoplasm of the cells, Î±-synuclein oligomer had been consistently detected in human cerebrospinal fluid (CSF) and blood plasma not only in diseased individuals but also those healthy. Moreover, Î±-synuclein was also demonstrated be released into the extracellular media by exocytosis and internalized into cells, implying that Î±-synuclein may not only functional intracellularly .
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In Î±-synuclein transgenic models, Î±-synuclein aggregation is always related to neuronal degeneration either by loss of function or death . Acknowledged from several imaging techniques, in the context of a living cells Î±-synuclein can form small oligomers, which may be associated with cytotoxicity. The formation of Î±-synuclein oligomers is linked to cell membrane damage and result in dysfunction of multiple cellular systems.
Since Î±-synuclein also may an important role in mediating both mitochondrial function and damage, cells which are Î±-synuclein overexpressing tend to display multiple markers of mitochondrial dysfunction, including increased protein oxidation, increased reactive oxygen species (ROS) production, loss of mitochondrial membrane potential and reduced Complex I activity . Research shows that Î±-synuclein is localized in the inner membrane of mitochondria 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 have 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. As before, Î±-synuclein causes mitochondrial dysfunction, which causes increased generation or ROX, which negatively impacts Î±-synuclein, thus causing increasing cycles of neuronal stress and impaired function, eventually leading to death the neuron and destruction of that nerve pathway. While Î±-synuclein is known to interact directly with lipid membrane, especially the vesicles . It has been also been found that several genes that deal with metabolism of lipid play a role in Î±-synuclein toxicity by yeast genome screening. Lipid metabolism is common to both cellular and mitochondrial membranes and may bear closer examination. 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 addition, a prion-like spread of Î±-synuclein aggregates has been recently proposed to contribute to the propagation of Lewy bodies throughout the nervous system during progression of PD, suggesting that the metabolism of extracellular Î±-synuclein might play a key role in the pathogenesis of PD . In the normal brain, Î±-synuclein immunostaining reveals a diffuse pattern of reactivity throughout the neuropil that would be consistent with a predominantly synaptic localization. Immunostaining is using in the location study of Î±-synuclein. However, in PD brains, Î±-synuclein antibodies have extremely high sensitivity stain LBs and LNs. For the high sensitivity, Î±-synuclein staining is used more widely instead of the less specific staining by the dye eosin. Although it has been proved Î±-synuclein has a close relation with PD, it is demonstrated that increased production of some specific biological molecules, like heat shock proteins (Hsps) can prevent toxic effects of Î±-synuclein in yeast and in flies .
1.3 The Introduction of Yeast
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 simpler and more fully-understood genome, but also because it is easy to analyze an eukaryotic organism given that the availability of genetic banks of diploid and haploid gene deletion screens . As a powerful genetic tool, budding yeast is adopted to reveal protein misfolding processes underlying human diseases , including Huntington's disease , prion disease , and amyotrophic lateral sclerosis as reported before where single gene deletion stain banks in diploid and haploid strains have proved central to elucidation of these various biological processes in yeast. Full understanding of these biological systems and processes in human cells and mammalian model animals obviously has lagged behind the understanding that has occurred in these powerful yeast systems.
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 a unicellular with a scalable system, it enables high throughput genetic and small-molecule screens. Apart from the advantages we mentioned above, most important part is its genetic tractability which enables its DNA to be transformed quite easily, at the same time homologous recombination is efficient . 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 contribute to neurodegenerative. 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 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 for human disease research: the model has to be relevant to human disease and easy to be analyzed, which are possessed by yeast and enables yeast to be 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 easily transformed genome allowing for either the addition or deletion of genes through homologous recombination, grows in a highly reproducible and genetically stable way and short generation time . Genomic homology is a phenomenon reported by several articles. At least 60% of yeast genes are shown robust human homologues or at least one conserved domain with human genes statistically . In addition, a close yeast homologue can be found among more than 25% of known human disease genes , 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 neurodegenerative, 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 intimately associated by multiple lines of evidence. Thanks to the conservation of the cellular protein quality system, the study of these processes are readily studied in the yeast genetic systems .
In yeast, the same as in mammalian cells, the central organelle for the production of reactive oxygen species (ROS) which is heavily implicated in neurodegeneration are the mitochondria as mentioned previously. The fermentative growth ability of yeast allows for 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 . The pathway proteins are translocated from the endoplasmic reticulum (ER) to the Golgi complex and then trafficked in vesicles to the plasma membrane, is of particular importance in 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 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 a significant advantage over transformed or cancer derived immortal mammalian cell culture lines, and these characteristics are particularly necessary in a model system for initial screening for agents for therapeutic treatment of neurodegenerative diseases. Yeast phenotypes can be screened based on genome-wide deletion, reduced expression or overexpression libraries. Depending on whether a 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 . Clear yeast homologues exist for many genes associated with neurodegeneration. This modeling approach has already been proved to be highly productive .
1.4 The Introduction of High-throughput screening
Originated from natural product screening just for pharmaceutical, biological, biotechnological research and development 30 years ago, High-Throughput Screening (HTS) is a scientific method of experimentation particularly used in the process of drug discovery nowadays. The destination is to obtain a high efficiency by conducting copious of parallel biological experiments or tests in parallel using automation and miniaturization techniques, with the use of robotics, liquid handling devices, sensitive detectors, and data processing and control software . 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 the further screen against other targets.
A compound with a desired result in a high-throughput screening usually called hits, which 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 provide 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 inhibit 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 have the activity in preventing or reducing 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
2.1.1 Yeast Strains
Three Î±-synuclein overexpressing yeast strains from Dr. Susan Lindquist's lab were studied in the experiment: Yeast strain W303 is the parent strain that served as 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 these one 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 as listed below.
Table Genotypes of yeast strains W303, W303-CFP, W303-YFP and W303-CFP+YFP
MATa can1-100 his3-11, 15 leu2-3, 112 trp1-1 ura3-1 ade2-1 pRS306Gal pRS304Gal
MATa can1-100 his3-11, 15 leu2-3, 112 trp1-1 ura3-1 ade2-1 pRS306Gal-Î±-syn-CFP
MATa can1-100 his3-11, 15 leu2-3,112 trp1-1 ura3-1 ade2-1 pRS304Gal-Î±-syn-YFP
MATa can1-100 his3-11, 15 leu2-3,112 trp1-1 ura3-1 ade2-1 pRS306Gal-Î±-syn-CFP pRS304Gal-Î±Syn-YFP
The control strain W303 was constructed with two copies of empty vector integrated into Trp1 and Ura3 loci. Single integrated W303-CFP and W303-YFP strains were constructed by incorporating pRS306 and pRS304 vectors into the Ura3 and Trp1 loci of W303 respectively. As for 2X integrated W303-CFP+YFP strain, both pRS306 and pRS304 vectors were incorporated into the Ura3 and Trp1 loci of W303.
The gene structure of the yeast strains W303-CFP, W303-YFP and W303-CFP+YFP with galactose promoter to produce Î±-synuclein protein in this experiment is schematically shown below in Figure 2.
Figure Schematic diagram of the yeast gene for strains W303-DFP, W303-YFP and W303-CFP+YFP
Transgenetic Î±-synuclein overexpression yeast strains, marked with fluorescent proteins CFP and YFP were adopted in our experiment. The expression of Î±-synuclein is controlled by the GAL1 promoter: galactose promotes the expression of Î±-synuclein which can be prohibited by glucose in reverse. With the carbon source in the medium switched from raffinose to galactose, the transgene is induced and Î±-synuclein protein is produced, leading to the death of cells . The overall survival rate of cells increases with the presence of glucose compared to the galactose. W303-CFP and W303-YFP strains are single Î±-synuclein copy strains, with only one Î±-synuclein gene repeat, while W303-CFP+YFP strain with double Î±-synuclein copies, with double Î±-synuclein genes. In this circumstance, with the presence of galactose in the medium, W303-CFP+YFP strain was expected to have two folds of Î±-synuclein protein expression compared to individual W303-CFP strain and W303-YFP strain.
2.1.2 Yeast Media
We used three different yeast media in the experiment, SD-AA medium used for strains selection, YPR medium used for strains growing and YPR+ 0.5% Gal medium for activating the galactose induced Î±-synuclein gene during the screening. The recipes for SD-AA medium, YPR medium and YPR+ 0.5% Gal Medium are shown below:
Table The media used in the screening
Yeast Nitrogen Base
Yeast Nitrogen Base
Yeast Nitrogen Base
YPR+ 0.5% Gal Medium
The SD minus media contained complete supplement mixture minus some essential components like uracil (Ura) and tryptophane (Trp). Since the medium is devoid of essential components, prototrophs with self-produce Ura and Trp ability are able to grow, providing selection for plasmid-containing strains.
The YPR medium changed the sugar Dextrose (Glucose) in YPD medium to Raffinose. Given that Glucose served as an inhibitor of Î±-synuclein expression in this experiment, the strains where grown in a non-glucose containing medium and were cultured overnight at 30â„ƒ in YPR medium for screening in YPR medium in preparation for screening when glucose could be added if inhibition was required by the experimental designr
The YPR + 0.5% Galactose medium were used for inducing the production of Î±-synuclein protein. With the presence of galactose, the galactose-inducible promoter is able to promote the expression of the Î±-synuclein-CFP and Î±-synuclein-YFP genes.
2.1.4 Blueberry compounds for screening
The compounds from Dr. Rochet's lab, Dept. of Medicinal Chemistry and Molecular Pharmacology, Purdue University listed in Table 2 were screened to determine the protective capability of rescuing yeast cells from negative impact of Î±-synuclein protein. In the high-throughput screen, we tried to identify active compounds (hits) from this library and figure out the suitable concentration of the hits.
Table Compounds in blueberry library for high-throughput screening
Bb PAE (sugar-free polyphenol rich)
BbÂ Seph 1Â (100 % anthocyanins)
BbÂ Seph 2+3
BbÂ Seph 4
BbÂ Seph 5
Soy (Jim Simons)
The compounds 1 to 7 are crude extracts (e.g. blueberry extract) or fractions of the extracts (e.g. anthocyanin-rich fraction, etc) while compounds 8 to 17 are pure compounds. Seph 1, 2, 3, and 4 are fractions with different amounts of anthocyanins, whereas Seph 5 is a fraction enriched with proanthocyanidins as listed in the Table 3 below (confidential):
Table Anthocyanin and Chlorogenic acid Content of compounds 1-7
Chlo A mg/g
% in extract
PAE (sugar free ext)
Figure The concentrations of Anthocyanins and chlorogenic acid in compounds 1 to 7 (Unit: mg/g)
Samples 1-7 from Wild bluberries representing different extracts and fractions: from crude extract to 100 % anthocyanins (sample 4), or ~100 proanthocyanidin polymers (sample 7). Chlorogenic acid is also present in some of the natural product isolation fractions as indicated in the histogram of the Figure 3 above.
2.2.1 Yeast strain culturing
The strains were cultivated in 50ml centrifuge tubes (BD) overnight at 30â„ƒ, 110 rpm in 5 ml SD minus medium, W303-CFP strain in the SD-URA medium, W303-YFP strain in the SD-TRP medium, and W303-CFP+YFP strain in the SD-TRP-URA medium.
2.2.2 Frozen stock preparing
One single colony for each strain was used to ensure that the genotypes maintained the same for all the replications during the experiment. Frozen stocks were prepared in the following way: Single colonies were picked from the petri dish grew overnight in the SD minus medium. 0.54 ml 50% glycerol was first added to 0.96 ml culture suspension to reach 18% glycerol concentration, then allocated 100 Î¼l to each tube (15 tubes were used). The sample tubes were frozen at -80â„ƒ for further use.
2.2.3 Preparing plates for screening Yeast Cells
First, 50Î¼l of frozen Yeast culture was thawed and cultured in a 50 ml Falcon tube with 5ml YPR medium overnight, at 30â„ƒ and shaker-speed of 110rpm. Next the 200 Î¼l of yeast cell culture was diluted 100-fold (in YPR + 0.5% Galactose) to a final volume of 20 ml. This diluted yeast culture was then seeded into a 96-well plate, 120 Î¼l per well. Test-compounds were individually added to different wells in the 96-well plate. Using the Laboratory Automation Workstation (Biomek FX), the contents of the 96-well plate was transferred into a 384-well plate. The volume from each well of the 96-well plate was split and deposited into 4 separate wells (25 Î¼l per well) on the 384-well plate.
The 384-well plate was put in the stacker of an automatized Microplate Reading System (Molecular Devices). Once every hour for 48 hours, a robot would take out the plate from the stack and insert it into the micro-plate reader for quantification of OD at 600nm. The whole room was kept at 30 â„ƒ to optimize growing conditions for the yeast. The entire procedure can be illustrated by the figure below:
Figure Schematic procedure of Primary Screen
2.2.4 Monitoring Î±-synuclein-fluorescent protein expression by flow cytometry
The strains were grown in YPR + 0.5% Gal medium overnight to initiate the expression of fluorescent protein and Î±-synuclein fusion gene, BECKMAN COULTER flow cytometer (ADP Cyan) was used for fluorescent examination and data was analyzed by summit V4.3.02.
2.2.5 Investigating Cell survival under Galactose and Glucose
The purpose of this experiment was to compare the growth rate of the four different yeast strains in the presence of galactose and to deduce the effective concentration of galactose for subsequenct experiments. A 384-well screening plate containing all four strains was prepared by the laboratory robot (Biomek FX) and the yeast cells were cultured in YPR-medium containing either 0.1% and 0.5% galactose. The medium in other wells also supplemented with either 0.1% and 0.5% glucose to monitor the effect of glucose on the expected galactose-effect. As an internal control, yeast cells were cultured without galactose/glucose in column 1 and 2. After preparation, the plate was kept at 30â„ƒ for 48 hours and OD600 was monitored hourly by a microplate spectrophotometer system (Molecular Devices).
2.2.6 Master compound library preparation
The different blueberry compounds had to be tested in different concentrations to ascertain their efficacy. This section describes the preparation of 15 master plates with 5 different concentrations (undiluted, 2X, 4X, 8X and 16X-diluted) of blueberry compounds using the Biomek FX robot according to a previously defined dilution program:
One ml of 100-fold dilution of each substance in the blueberry-compound-library and three controls were first deposited in a 96-deep-well plate.
800 ul from each well in the 1st deep-well plate was transferred to a 2nd empty plate. This plate represents undiluted compounds.
400 ul from each well in the 2nd plate was transferred to a 3rd plate that already contained 400 ul of YPR+ 0.5% Gal media per well. Thus achieving a 2X dilution.
400 ul from each well in the 3rd plate was transferred to a 4th plate that already contained 400 ul of YPR+ 0.5% Gal media per well. Thus achieving a 4X dilution.
400 ul from each well in the 4th plate was transferred to a 5th plate that already contains 400 ul of YPR+ 0.5% Gal media per well. Thus achieving a 8X dilution.
400 ul from each well in the 5th plate was transferred to a 6th plate that already contains 400 ul of YPR+ 0.5% Gal media per well. Thus achieving a 16X dilution.
The end product of this dilution procedure is 5 plates containing (400ul per well) with 5 different concentrations of each blueberry compound (undiluted, 2X, 4X, 8X and 16X-diluted). Next the contents of each plate (400 ul/well) was split into 3 separate plates (120ul/well), thus yielding 3 master plates per concentration, altogether 15 plates. A schematic of this procedure is provided in Figure A2 in the Appendix.
2.2.7 High-throughput screening
In the high-throughput screen, the primary screen is in order to find hits from compound libraries, while the secondary screen could find out the suitable concentration of the hits.
Due to the limited time and the small blueberry library which just has 17 compounds, I combined the primary and secondary screen together. In this screen, all of the 17 compounds from the blueberry library were screened at 5 concentrations. All compounds were diluted by 100 folds as the highest concentration, and then 5 steps of 2-fold dilution to get the efficiency concentration of the compounds as well as the hits.
As a chemical activator of the heat shock response, Geldanamycin (GA) demonstrates the ability to inhibit Î±-synuclein-triggered apoptosis by inducing the expression of heat shock proteins. It inhibits the Hsp90-mediated conformational maturation or refolding reaction by binding to a conserved binding pocket in ATP-binding domain of Hsp90 , resulting in the degradation of Hsp90 substrates . It has the ability to protect the transgenic fruit flies and yeast from the deleterious effects of Î±-synuclein. Treatment with GA abolishes the ability of Î±-synuclein to induce reactive oxygen species (ROS) accumulation, which was used as a marker of apoptosis . In our experiment, GA was used as a positive control in the high-throughput screen.
Figure Plate Layout of 384-well plate, high-throughput screen
Medium: YPR + 0.5% Gal. Column1 and 2 were controls, Glucose and GA acted as positive controls, and DMSO served as a negative control. All of the four strains were treated with the 17 compounds from the Blueberry library and three controls, OD were measured every hour for 48 hours.
CHAPTER III RESULTS and DISCUSSION
3.1 Consummation of fluorescent protein production by Flow Cytometry
The yeast strains W303-CFP, W303-YFP and W303-CFP+YFP were constructed to produce the Î±-synuclein gene fused fluorescent protein CFP and YFP under control of the GAL1 promoter. Among the four yeast strains, W303 with empty vectors served as negative control in this experiment. The four yeast strains (strains W303ï¼ŒW303-CFP, W303-YFP and W303-CFP+YFP) were cultured with YPR+ 0.5% Gal medium overnight to enable the expression of fluorescent protein and Î±-synuclein fusion gene and the expression was monitored by detection of CFP and YFP fluorescence by flow cytometry.
Figure Detect fluorescent protein expression by flow cytometry for yeast strains W303 (A), W303-CFP (B), W303-YFP (C) and W303-CFP+YFP (D)
From the result of flow cytometry showed in Figure 6, 22075 cells of W303 strain were counted and 0.08% of them (20 cells) showed YFP like signal and 3.43% (758 cells) showed CFP like signal. As for W303-CFP strain, 28025 CFP positive cells formed 83.58% of 33748 counted cells, while there were 0.45% of cells (151) displayed YFP positive. In Figure 6C, 23957 YFP positive cells made up 92.5% of 25899 counted cells, and CFP positive cells is 1483 account for 5.73%. In 22157 counted W303-CFP+YFP cells, 17114 of them were YFP positive (77.24%) and 4627 were CFP positive (20.88%) but only 18.88% expressed both genes as it shows below in Table 5:
Table The expression of fluorescence by flow cytometry
From the result we obtained from flow cytometry in Table 5, only W303-CFP strain, W303-YFP strain and W303-CFP+YFP strain exhibited fluorescence, indicating that the fluorescent protein expressed with the treatment of galactose, while hardly any fluorescent protein was detected in W303.
The results of flow cytometry analysis confirmed that the fused Î±-synuclein genes were expressed in the presence of galactose. In the negative control, there were still 0.09% YFP signal like and 3.43% CFP signal like among the cells, this might be caused by cell aggregation, aggregated cells reflected more light that could be detected by flow cytometer, which is acceptable. Also, the 0.45% YFP signal like cells in W303-CFP strains which were acceptable. In W303-CFP+YFP cells, there were 77.24% YFP positive and 20.88% CFP positive while only 18.88% expressed both CFP and YFP fluorescence, which revealed that not all of the W303-CFP+YFP cells efficiency expressed the fused genes, and the level of Î±-synuclein protein produced by the strains integrated with double Î±-synuclein genes may not have been two-fold of that produced by single integrated Î±-synuclein strains.
3.2 Effects of Î±-synuclein expression on yeast growth curves
To detect if the Î±-synuclein protein is able to cause cell damage, we induced the expression of Î±-synuclein with galactose treatment. Reduction of yeast cell growth rate was expected to be detected in this experiment if Î±-synuclein protein has negative effect on cell growth, and the hypothesis was made that all three Î±-synuclein overexpressing strains treated with two concentrations of galacose had less growth rate than the control. In order to monitor the variation between experiments, eight 384-well plates were repeated, all of them displayed same situation.
The results confirmed that the Î±-synuclein genes in these strains were induced by galactose, which represents that with the treatment of galactose, Î±-synuclein protein expressed and finally result in the cell damage. Due to the prohibitive effect of glucose against GAL1 promoter, glucose treated strains served as the positive control in this assay. The yeast cells turned out to have a higher growth rate with the presence of glucose in the medium. Strains grown with raffinose as the only carbon source served as controls.
Figure Growth curves under the treatment of 2% Raffinose screened at 30 â„ƒ for 48 hours with X-axis showing time (hr) and Y-axis showing optical density Log(OD)
Figure 7 above is the growth curve with raffionse as the only carbon source, severed as a control. All of the four strains had normal growth curves. W303-CFP strain displayed the fasted growth rate among the four cell lines with the log phase started at 3rd hour and finished at 22nd hour. The wild type (i.e. W303) is almost the same with W303-CFP+YFP strain with 7 hours lag phase and got into the stationary phase after 25 hours. W303-YFP strain was the weakest one among the four strains, the lag phases finished after about 8 hours, and reached the stationary phase after about 26 hours. After 48 hours experiment, W303-CFP got the highest Log (OD), and then W303 and W303-CFP+YFP, while the W303-YFP was the lowest one.
Figure Growth curves under the treatment of 0.5% (A) and 0.1% (B) Galactose in the medium screened at 30 â„ƒ for 48 hours with X-axis showing time (hr), Y-axis showing optical density Log (OD)
As the figure 8A above shows, when treated with 0.5% galactose, it is obvious that the growth rates of Î±-synuclein overexpression strains were significantly slower than the control W303 strain. W303 strain's log phase started at 8th hour and ended at 23 hours. Compared to the control experiment, strains with single Î±-synuclein gene incorportated (W303-CFP and W303-YFP strains) showed a relatively long time of lag phase: strain W303-CFP stepped into log phase after 18 hours and the stationary phase from the 37th hour and for strain W303-YFP it even took 20 hours for entering into log phase and didn't got into the stationary phase in the 48 hours experiment. In addition, the combination of CFP and YFP Î±-synuclein genes displayed a severe negative impact on yeast cell growth: W303-CFP+YFP strain started to grew after 44 hours.
Figure 8B is the growth rates of cells treated with 0.1% galactose. W303 strain and W303-CFP strain grew quiet similar: most parts of their log phases overlapped, although W303-CFP strain started to divide 4 hours before W303 at the 4th hour, they got into the stationary phase almost at the same time after 27 hours of the experiment. Nevertheless it was still faster than W303, the W303-CFP strain's log phase was still delayed compared to its growth rate with raffinose (control). Although the W303-YFP strain has only one copy of the Î±-synuclein gene as W303-CFP strain, it grew much lower than W303-CFP strain. The yeast cells spent 11 hours to adapt themselves to the growth condition and entered the stationary phase at 35th hour. As for the W303-CFP+YFP strain, with double integrated Î±-synuclein gene, cannot grow normally: after 23 hours of the screen, it began to grow slowly and didn't get into the stationary phase after 48 hours experiment.
Figure Growth curves under the treatment of 0.5% (A) and 0.1% (B) Glucose screened at 30 â„ƒ for 48 hours with X-axis showing time (hr), Y-axis showing optical density Log(OD)
Figure 9A above shows that with the prohibit effect by glucose on GAL1 in this experiment, all of the four strains revealed normal growth curves. Strain W303-CFP was the first one adapt to the environment in 4 hours, got into the stationary phase around 17th hour. After 6 hours lag phase, the high speed growth of W303 strain kept to 24th hour. The growth curve of the double mutate was between YFP and W303, but quite similar to W303 at the beginning. The log phase started at 6th hour and ended at 20th hour. W303-YFP strain was the slowest one: got into the log phase after 9 hours and reached stationary phase after 30 hours. Compared to the strains in galactose, the strains treated with glucose showed obvious protective effect to restore the cells to normal growth rate although the protective effect varies according to different yeast cell lines.
Figure 9B revealed the growth curves with 0.1% glucose, all of the four strains grew normally. W303-CFP strain was still maintained fastest growth rate compared to other yeast strains: high speed growth continued from 4th hour to 16th hour. Strains W303-CFP+YFP and W303 still overlapped at the beginning, got into the log phase at 6th hour and entered the stationary phase at around 23rd hour. W303-YFP strain, the weakest one, end up with the lag phase after 9 hours screen and entered into stationary phase after 23 hours.
Similar results were got in the eight repeated plates, which confirmed that the method and models were stable, they are suitable to be applied to the following experiments and even more compounds selection. From the results of the galactose and glucose treatment experiment, it's obvious that the Î±-synuclein protein significantly impaired growth of yeast cells, which is in agreement with numerous previous studies suggested that the overexpression of Î±-synuclein negatively impacts model organisms such as Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans . When the concentration of galactose was 0.5% in the medium, Î±-synuclein was more effectively induced than at 0.1% galactose as we showed above, therefore, 0.5% galactose was used in all the following experiments to test the effects of compounds to rescue cells suffering from Î±-synuclein cytotoxicity.
These results further proved that when the Î±-synuclein gene was overexpressed, some of the cells maintained viability although the growth rate was affected. And cell growth was further reduced when Î±-synuclein protein was more expressed by the double fusion genes (W303-CFP+YFP). Previous study proved that the neurotoxic potential of Î±-synuclein is mainly determined by its protein levels .
3.3 Screening the blueberry-library compounds for protect against Î±-synuclein induced cytotoxicity
It was show above that Î±-synuclein has a detrimental effect on yeast cell growth rate. We could therefore apply the four yeast strains to screen for compounds that protect cells against this toxicity. From the difference of the curves that we calculated using R software, there are several compounds showing distinctive effect on rescuing cells from Î±-synuclein cytotoxicity. Bb PAE (sugar-free polyphenol rich) (Compound 3) and BbÂ Seph 5 (compound 7) showed excellent hits as expected, glucose and GA also displayed effective positive controls as in Figure 11.
Figure Growth curve of four strains in the high-thoughtput screen at 30 â„ƒ for 48 hours: the growth curve with the YPR+ 0.5% Gal medium (A), growth curve with glucose exposure (B) and GA (E) in the YPR+0.5% Gal medium, and growth curve after the addition of compounds 3 (C) and 7 (D) to the YPR+ 0.5% Gal medium.
In the Figure 11 above, strains expressing the Î±-synuclein gene prohibit cell growth with treatment of galactose while the W303 grew normally. Glucose reduced the Î±-synuclein effect in all three other strains (i.e. W303-CFP, W303-YFP and W303-CFP+YFP strains) we used. Effective rescuing was also observed when compounds 3 and 7 were added to the medium.
Figure OD curves of W303-CFP+YFP strain on plate 1 in the high-throughput screen at 30 â„ƒ for 48 hours. Cells were grown in YPR+0.5% Gal media, compounds were added to the medium with X-axis showing time (hr) and Y-axis showing optical density Log(OD).
The W303-CFP+YFP strain exhibited the strongest cytotoxicity effects of Î±-synuclein, presumebly because it expressed two copies of Î±-synuclein fusion genes. An overview of the protective effect of compounds from the blueberry library on this strain is shown in Figure 12. With 100 Âµg/ml treatments of compounds 1, 3, 4, 5 and 7, the W303-CFP+YFP strain revealed varying degrees of rescue. The most effective compound is compound 7 the final OD reached two third of the control experiment, which treated with glucose. For the treatment with compounds 1, 3, 4 and 5, various levels of cell growth was observed, not as effective as compound 7.
Previous researches confirmed that dietary interventions might provide an effective strategy for preventing neurodegenerative disorders . A Blueberry enriched diet provided significant protection against these decrements in performance and enhanced dopamine recovery in animal .Greater intakes of berries were strongly associated with a lower risk of developing PD . Neuroprotective activity of strawberries was tested in vitro on PC12 cells treated with H2O2 . Why berries are effective is not clear yet, but we have demonstrated that certain compounds are very effective in rescuing cells from Î±-synuclein. In this project, the compounds extracted from blueberries were applied to rescue the yeast cells from the Î±-synuclein overexpression condition.
The compounds with different amounts of anthocyanins were detected to be protective, and the fraction enriched with proanthocyanidins (Compound 7) was proved to be the most effective one, which means that there were at least two components in blueberry were effective and the displayed significant positive effect.
The mechanism involved in the protection is still not clear but a diet enriched in Blueberries might provide protection against neurodegenerative processes due to cellular stresses such as oxidative stressors .
The antioxidative activities of proanthocyanidins were found to be much stronger than vitamin C or vitamin E in aqueous systems . The intake of proanthocyanidins, which are naturally occurring antioxidants, increases the resistance of blood plasma against oxidative stress .
Figure OD curves of W303-CFP+YFP strain on plate 2 (A) and 3 (B) in the high-throughput screen at 30 â„ƒ for 48 hours. Cells were grown in YPR+0.5% Gal media, compounds were added to the medium with X-axis showing time (hr) and Y-axis showing optical density Log(OD).
From the Figure 12 above, even the concentrations of the compounds decreased to 50 ug/ml (Figure 12A), effective rescue could still be observed. As long as the concentrations decreased to 25 ug/ml (As Figure 12B indicated above) however, the effects were not significant anymore. Even the most effective compound 7 was not able to significantly rescue the W303-CFP+YFP strain. From this result, more concentration gradients between 25 ug/ml to 50 ug/ml are required to figure out the lowest effective concentration.
On Plate 1, at time point 1 of the high-throughput screen, the OD of some treatments reached a peak and then reduced, especially in compounds 3, 4, 5 and 6. But the same situation did not appear in the treatment with lower concentrations like 25 Âµg/ml of compounds. This might be explained by the color changes of anthocyanins, which is very abundant in these compounds (Compounds 3, 4, 5 and 6). Anthocyanins may appear red, purple, or blue depending on different pH values. When the compounds were added to the medium, pH of the medium changed with the consumption of YPR, leading to the color change of the anthocyanins. OD measured under 600 nm increased when anthocyanins became purple, with the acidic materials produced by yeasts due to metabolism, the pH of medium decreased, and anthocyanins changed back to red, cause OD to decrease.
CHAPTER IV CONCLUSIONS
In the present study, we did yeast screening for the expression of Î±-synuclein with induction of galactose. The yeast strains W303-CFP, W303-YFP and W303-CFP+YFP expressed Î±-synuclein and fluorescent protein which were detected by the flow cytometry after induction with galactose. Î±-synuclein protein expression was detrimental to the yeast cells, as confirmed by the growth curve in presence of galactose. In contrary, glucose as a suppressor played an important role in protecting cells from Î±-synuclein toxicity, presumably by preventing activity of the GAL1 promoter and expression of the Î±-synuclein.
Blueberry enriched food reduced the PD risk in a 20-year follow-up experiment. The blueberry compound library was screened to search for effective rescue compounds in the Î±-synuclein overexpression yeast cultures.
In the rescue screening, several positive results were obtained. Bb PAE extract (sugar-free polyphenol rich) (compound 3) and BbÂ Seph 5 fraction (compound 7) from the blueberry compound library illustrated effectively protective effects on the cells, rescued the cells from growth inhibition, which was concluded from the growth curve with galactose treatment compared to the controls. Besides the compounds 3 and 7, Bb-crude (compounds 1), BbÂ Seph 1Â (100 % anthocyanins) (compound 4) and BbÂ Seph 2+3 (compound 5) showed less effective. Several repeats were screened to confirm the effects of the compounds. BbÂ Seph 5 (compound 7) illustrated highly efficiency protective effect against the Î±-synuclein toxicity.
When the concentrations were 50 ug/ml, the compounds are effective, but when the concentration decreased to 25 ug/ml, the rescue effectively redued. More experiments should be done to find out the lowest effective concentration.
Our results suggested that proanthocyanidins, rich in BbÂ Seph 5 (compound 7), may play an important role in protection against Î±-synuclein toxicity, which coincides with the results from previous research.
CHAPTER V FUTURE WORKS
Based on the results we acquired in the yeast screen which showing that the compounds from blueberries have significant protective effects on yeast cells, we presume that the compounds are capable of protecting the human cells from Parkinson's disease. A series of experiments could be designed to test the hypothesis: Treating the SHSY5Y cells with Rotenone to imitate the symptoms of Parkinson's disease, and making attempt to rescue them by the blueberry compounds. High-content screening will be used in the assay of the plate. Imaging cytometry techniques will be implemented to simultaneously monitor several parameters extracted from quantitative analysis of biomarker intensity and distribution.
With the characteristics of dopaminergic neurons we discussed above, SH-SY5Y cell line was proved to be an ideal cell model for further PD research in our research. As a comparatively homogenous neuroblast-like cell line, SH-SY5Y cell line is a thrice cloned subline of SK-N-SH cells which were originally derived from a bone marrow biopsy of a metastatic neuroblastoma site in a four year-old female in 1970's. As this cell line possesses many biochemical and functional properties of neurons, it has been broadly used as model of neurons since the early 1980's. In addition, SH-SY5Y cells possess the capability of proliferating in culture for long periods without contamination, which is required by in vitro cell model development. In conclusion, due to the advantages and characteristics SH-SY5Y cell line has, it has been applied to experimental neurological studies, including analysis of neuronal differentiation, metabolism, and function related to neurodegenerative and neuroadaptive processes, neurotoxicity, and neuroprotection .
Multi-parametric high-content screening (mp-HCS) of mitochondrial toxicity holds promise as a lead in-vitro strategy for drug testing and safety evaluations. In the future steps of this study, an mp-HCS and multi-parametric data analysis scheme for assessing cell responses will be developed for Parkinsonism inducing.
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