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- Title
- ALPHA-SYNUCLEIN: INSIGHT INTO THE HALLMARK OF PARKINSON'S DISEASE AS A TARGET FOR QUANTITATIVE MOLECULAR DIAGNOSTICS AND THERAPEUTICS.
- Creator
-
Evangelista, Baggio A, Kim, Yoon-Seong, University of Central Florida
- Abstract / Description
-
Parkinson's disease (PD) is the second-most common neurodegenerative disease after Alzheimer's disease. With 500,000 individuals currently living with Parkinson's and nearly 60,000 new cases diagnosed each year, this disease causes significant financial burden on the healthcare system - amassing to annual expenditures totaling 200 billion dollars; predicted to increase through 2050. The disease phenotype is characterized by a combination of a resting tremor, bradykinesia, muscular rigidity,...
Show moreParkinson's disease (PD) is the second-most common neurodegenerative disease after Alzheimer's disease. With 500,000 individuals currently living with Parkinson's and nearly 60,000 new cases diagnosed each year, this disease causes significant financial burden on the healthcare system - amassing to annual expenditures totaling 200 billion dollars; predicted to increase through 2050. The disease phenotype is characterized by a combination of a resting tremor, bradykinesia, muscular rigidity, and depression due to dopaminergic neuronal death in the midbrain. The cause of the neurotoxicity has been largely discussed, with strong evidence suggesting that the protein, alpha-Synuclein, is a key factor. Under native conditions, alpha-Synuclein can be found localized at synaptic terminals where it is hypothesized to be involved in vesicle trafficking and recycling. However, its biochemical profile reveals a hydrophobic region that, once subjected to insult, initiates an aggregation cascade. Oligomeric species-products of the aggregation cascade-demonstrate marked neurotoxicity in dopaminergic neurons and illustrate migratory potential to neighboring healthy neurons, thereby contributing to progressive neurodegeneration. The current golden standard for PD diagnostics is a highly qualitative system involving a process-by-elimination with accuracy that is contingent upon physician experience. This, and a lack of standardized clinical testing procedures, lends to a 25% misdiagnosis rate. Even under circumstances of an accurate PD diagnosis, the only treatment options are pharmacologics that have a wide range of adverse side effects and ultimately contribute to systemic metabolic dysfunction. Thus, the research presented in this thesis seeks to overcome these current challenges by providing (1) a quantitative diagnostic platform and (2) a biomolecular therapeutic, towards oligomeric alpha-Synuclein. Aim 1: serves as a proof-of-concept for the use of catalytic nucleic acid moieties, deoxyribozymes and aptamers, to quantify alpha-Synuclein in a novel manner and explore the ability to detect oligomeric cytotoxic species. The cost-effective nature of these sensors allows for continued optimization. Aim 2: serves to establish a potential therapy that can abrogate alpha-synuclein oligomerization and toxicity through use of a modified Protein Disulfide Isomerase (PDI) peptide when introduced to live cells treated to simulate pre-parkinsonian pathology.
Show less - Date Issued
- 2017
- Identifier
- CFH2000188, ucf:46024
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000188
- Title
- Implication of alpha-synuclein transcriptional regulation and mutagenesis in the pathogenesis of sporadic Parkinson's disease.
- Creator
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Basu, Sambuddha, Kim, Yoon-Seong, King, Stephen, Estevez, Alvaro, Altomare, Deborah, University of Central Florida
- Abstract / Description
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Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by selective loss of dopaminergic neurons (DA neurons) from the substantia nigra (SN) of the mid-brain. PD is classically associated with cytoplasmic inclusion of aggregated proteins called Lewy bodies. alpha-synuclein (?-SYN) coded by the gene SNCA, is one of the major components of Lewy body and neurite along with several other proteins like ubiquitin, neurofilament to name a few. PD is broadly categorized...
Show moreParkinson's disease (PD) is an age-related neurodegenerative disorder characterized by selective loss of dopaminergic neurons (DA neurons) from the substantia nigra (SN) of the mid-brain. PD is classically associated with cytoplasmic inclusion of aggregated proteins called Lewy bodies. alpha-synuclein (?-SYN) coded by the gene SNCA, is one of the major components of Lewy body and neurite along with several other proteins like ubiquitin, neurofilament to name a few. PD is broadly categorized into two groups based on their incidence of occurrence. First is the familial form that occurs due to known genetic aberrations like mutation, gene duplication/triplication in important PD associated gene like SNCA which in turn leads to early-onset PD (EOPD). Second is the late-onset idiopathic or sporadic form, whose origin of occurrence is often unknown. Interestingly, more than 90%-95% of reported PD cases belong to the latter category. Although, the familial and the idiopathic form of PD are different in their respective cause of occurrence, aggregation of ?-SYN into Lewy body is a common pathologic hallmark seen in both. Aggregation of ?-SYN in turn is strongly implicated by the transcriptional upregulation of the gene as seen in both familial forms as well as idiopathic forms. In this thesis, we first describe the designing and functioning of a novel tool to monitor real-time SNCA transcription in Human Embryonic Kidney (HEK) 293T cells. In the next part, we shed light into a novel transcriptional deregulation phenomenon called transcriptional mutagenesis, which leads to accelerated aggregation of ?-SYN as seen in sporadic PD. In brief, the focus of this work is to highlight the importance of transcriptional regulation of SNCA gene, through development of a tool and a mechanism affecting the fidelity of transcription under pathologic condition. In the first study, we developed a stable cell line in HEK293T cells in which ?-SYN was tagged with Nanoluc luciferase reporter using CRISPR/Cas9-mediated genome editing. Nanoluc is a small stable reporter of 19KDa size, which is 150 fold brighter compared to firefly and Renilla luciferase, thus making it a very good candidate for endogenous monitoring of gene regulations. We successfully integrated the Nanoluc at the 3'end of the SNCA before the stop codon. Successful integration of the Nanoluc was demonstrated by the fusion ?-SYN protein containing the Nanoluc. This allowed efficient monitoring of ?-SYN transcription keeping its native epigenetic landscape unperturbed which was otherwise difficult using exogenous luciferase reporter assays. The Nanoluc activity monitored by a simple two-step assay faithfully reflected the endogenous deregulation of SNCA following treatment with different drugs including epigenetic modulators and dopamine which were already known to up-regulate SNCA transcription. Interestingly, use of exogenous promoter-reporter assays (firefly luciferase assays) failed to reproduce the similar outcomes. In fact, exogenous system showed contradictory results in terms of the ?-SYN regulation which aroused from spurious effects of the drug on the reporter system. To our knowledge, this is the first report showing endogenous monitoring of ?-SYN transcription, thus making it an efficient drug screening tool that can be very effectively used for therapeutic intervention in PD. In the next study, we investigated the effect of oxidative DNA damage in the form of 8-hydroxy-2-deoxyguanosine (8-oxodG, oxidized guanine) on aggregation of ?-SYN through a novel phenomenon called transcriptional mutagenesis. It is already known that 8-oxodG is repaired by a specific component of the base excision repair machinery of the cell called 8-oxodG-DNA glycosylase 1 (OGG1). If left unrepaired, 8-oxodG can lead to misincorporation of adenine instead of cytosine (C?A transversion) in the synthesized mRNA during transcription for post-mitotic cells like neurons. This phenomenon is called transcriptional mutagenesis (TM) and can generate novel mutant variants of any functional protein. ?-SYN, which is implicated very strongly in the pathogenesis of PD, has been shown to become aggregation prone by specific point mutation. Previous studies have shown that certain point mutations can make ?-SYN more prone to aggregation and can affect the aggregation of the parental protein as a template directed misfolding mechanism. We used SNCA as a model gene and predicted the generation of forty-three different positions that can be mutated by the TM event. We investigated the generation of three out of the forty-three possible TM mutants from the SN of post-mortem PD and age-matched control brain cohorts based on their potential to aggregate as predicted by aggregation prediction software TANGO. The three mutants were Serine42Tyrosine (S42Y), Alanine53Glutamate (A53E) and Serine129Tyrosine (S129Y). We confirmed the presence of all the three mutant ?-SYN (S42Y, A53E and S129Y) in SNCA mRNA from the SN of human post-mortem PD brain using a PCR-based detection technique. As expected, analysis of the overall distribution of the three mutants showed a higher rate of occurance in the PD cohort compared to the age-matched controls. Sequencing genomic DNA of the same PD sample from the same region of ?-SYN revealed no mutations at the genomic DNA level, thus implying its generation during transcription. Although we could detect the presence of S42Y, A53E and S129Y ?-SYN in the cohort of PD patients, we focused to analyse the contribution of S42Y towards the aggregation of wild-type (WT) ?-SYN parental protein based on its higher potential to aggregate. By using cell-based biochemical and recombinant protein assays, we saw that S42Y-?-SYN can accelerate the aggregation process involving the WT protein even when present in significantly lower proportion (100 times less compared to the WT). Importantly, we developed antibody to specifically detect the S42Y ?-SYN in human PD cohort. Immunohistochemical analysis of serial post-mortem PD brain sections with Hematoxylin and Eosin staining (H(&)E), anti-ubiquitin staining and anti-S42Y ?-SYN staining, showed Lewy bodies that stained positively with S42Y ? -SYN. To our knowledge, this is the first report about TM related mutations of ?-SYN in Parkinson's disease and their role in the pathogenesis.
Show less - Date Issued
- 2017
- Identifier
- CFE0006719, ucf:51882
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006719
- Title
- USING THE YEAST TWO-HYBRID SYSTEM TO DETERMINE THE FUNCTION OF PARKIN E3 UBIQUITIN LIGASE.
- Creator
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Nguyen, Vanessa, Zervos, Antonis, University of Central Florida
- Abstract / Description
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Parkin is a cytosolic E3 ubiquitin ligase that is recruited to the mitochondria during cellular stress and has been suggested to be involved in a variety of biological processes such as mitophagy. The recruitment of Parkin (PARK2) to the mitochondria is dependent upon the kinase activity and the accumulation of PINK1 on damaged mitochondria. Mutations in either PINK1 or Parkin genes disrupt this protective pathway and lead to the accumulation of damaged mitochondria. From a clinical...
Show moreParkin is a cytosolic E3 ubiquitin ligase that is recruited to the mitochondria during cellular stress and has been suggested to be involved in a variety of biological processes such as mitophagy. The recruitment of Parkin (PARK2) to the mitochondria is dependent upon the kinase activity and the accumulation of PINK1 on damaged mitochondria. Mutations in either PINK1 or Parkin genes disrupt this protective pathway and lead to the accumulation of damaged mitochondria. From a clinical standpoint, mutations in the PARK2 gene have been associated with the progression and onset of autosomal recessive juvenile parkinsonism. Without the presence of a quality control system such as that of the PINK1/Parkin pathway, the accumulation of damaged mitochondria could lead to increased levels of oxidative stress, a decrease in ATP, and the progression towards cellular death. However, many of the details regarding the mechanism of Parkin-mediated ubiquitination and its involvement in mitophagy are not fully established. The intent of this thesis is to further explore the function of Parkin by utilizing the yeast-two hybrid system to identify novel Parkin interactors/substrates. A HeLa (cervical cell carcinoma) cDNA library was screened using Parkin124-465 as the "bait" protein. From this screening, six positive Parkin interactors were isolated and characterized. Using this approach it is possible to gain a better understanding of the function of Parkin in regulating cellular processes such as mitophagy.
Show less - Date Issued
- 2014
- Identifier
- CFH0004679, ucf:45269
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004679
- Title
- Mechanisms of Alpha-Synuclein-Induced Neurodegenertaion in Parkinson's Disease and Stroke.
- Creator
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Belal, Cherine, Chan, Sic, Ebert, Steven, Self, William, Teter, Kenneth, University of Central Florida
- Abstract / Description
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Parkinson's disease (PD) is a debilitating neurodegenerative disorder affecting one million Americans. Despite its social and economic impact, the pathological cascades that lead to neuron dysfunction and degeneration in PD are poorly understood. Endoplasmic reticulum (ER) stress has been implicated as an initiator or contributing factor in neurodegenerative diseases including PD. The ER is an organelle central to protein folding and intracellular Ca2+ homeostasis. Perturbations of these...
Show moreParkinson's disease (PD) is a debilitating neurodegenerative disorder affecting one million Americans. Despite its social and economic impact, the pathological cascades that lead to neuron dysfunction and degeneration in PD are poorly understood. Endoplasmic reticulum (ER) stress has been implicated as an initiator or contributing factor in neurodegenerative diseases including PD. The ER is an organelle central to protein folding and intracellular Ca2+ homeostasis. Perturbations of these functions result in ER stress and upregulation of ER stress proteins, of which some have been implicated in counteracting ER stress-induced cell death. The mechanisms that lead to ER stress and how ER stress proteins contribute to the degenerative cascades remain unclear but their understanding is critical to devising effective therapies for PD. Both the accumulation of mutant a-synuclein (aSyn), which causes an inherited form of PD, and the inhibition of mitochondrial complex I function by PD-inducing neurotoxin lead to ER stress. The critical involvement of ER stress in experimental models of PD supports its potential relevance to PD pathogenesis and led us to test the hypothesis whether the homocysteine-inducible ER protein (Herp), an ubiquitin-like domain (UBD) containing ER-resident protein, can counteract mutant Alpha Syn- and neurotoxin- induced pathological cascades.
Show less - Date Issued
- 2011
- Identifier
- CFE0004470, ucf:49310
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004470