Browsing by Subject "Alpha-synuclein"
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Item Development of High-Throughput and High-Content Analysis Assays for Neurodegeneration-Related Intrinsically Disorderd Proteins(2020) Nathan, NoahWe developed a FRET-based protein-protein biosensor of Fused in Sarcoma (FUS), an Amyotrophic Lateral Sclerosis and Frontotemporal Dementia related protein. The FUS biosensor had a robust signal, yielding a FRET efficiency of 7.61% with low signal to noise ratio. Based on high-throughput FRET measurements, we determined a standard deviation of 0.0158 ns for the donor/acceptor fluorescent lifetime. In future drug screens for compounds that modulate FUS aggregation, the threshold for hits will be set at 2.45 ± 0.0474 ns (3 SD). In addition, we implemented a MATLAB script that quantifies the ratio of cytoplasmic to nuclear FUS-rich stress granules from fluorescent images of FUS-GFP-expressing N2a cells. We showed that sorbitol, which has been shown to cause FUS mislocalization via hypertonic stress, caused a shift in the cytoplasmic to nuclear ratio of FUS as compared to untreated cells. We implemented a second MATLAB automated algorithm that quantifies total neurite outgrowth from neurospheres expressing the Parkinson's disease-related protein alpha-synuclein. We found that the mutant aSyn A53T caused a reduction of 41% in total neurites as compared to WT aSyn-expressing neurospheres. This result was validated by counting neurites manually with ImageJ, which yielded a reduction in neurites of 49%.Item Soluble alpha-synuclein oligomers are associated with reduced synapsin expression and enhanced cognitive decline in Alzheimer's disease(2014-02) Greimel, Susan JeanWe recently proposed that soluble, intraneuronal alpha-synuclein (alpha-Syn) might modulate Alzheimer's disease (AD) pathophysiology in the absence of Lewy body (LB) pathology. With mounting evidence indicating that oligomeric forms of aggregation-prone proteins such as A-beta, tau and alpha-Syn may be the major bioactive deleterious agents involved in AD, frontotemporal dementia and Parkinson's disease, we sought to identify the nature of the soluble alpha-Syn species elevated in AD and to determine the relative contribution of soluble alpha-Syn oligomers to AD-associated cognitive deficits. Using enzyme-linked immunosorbent assays designed to detect oligomeric alpha-Syn in our well-characterized human cohort, we found elevated levels of soluble alpha-Syn oligomers (o-alpha-Syn) in AD brains compared to aged-matched controls in the absence of LB cytopathology. Upon finical measurements of soluble alpha-Syn in subjects with AD, we not only detected 2 forms of monomeric alpha-Syn but also apparent multimers of each monomer. Unexpectedly, only a subset of soluble o-alpha-Syn species was elevated intracellularly while extracellular o-alpha-Syn remained unchanged. Multivariate analyses revealed that the respective abundance of selective low molecular weight o-alpha-Syn was associated with cognitive deficits in multiple domains. Finally, we found that elevating o-alpha-Syn in an AD mouse model triggered a selective decrease in synapsins and exacerbated A-beta-induced cognitive deficits. Altogether, our data support differential roles for soluble, intraneuronal alpha-Syn oligomers in Alzheimer's disease, which could extend to other synucleinopathies.Item Targeting Pathological Protein Interactions in Drug Discovery(2021-07) Young, MalaneyThe interaction of proteins with themselves or other molecules is essential to biological function. The dysregulation of normal protein structure, folding, and interactions forms the basis of many human diseases. Elucidating the protein interactions which underlie various disease states is crucial to understanding disease progression and identifying protein targets for therapeutic development. This dissertation focuses on targeting pathological protein interactions in cancer, non-alcoholic fatty liver disease (NAFLD), and neurodegenerative disorders. The tools developed in this dissertation are not only relevant to these specific disease states, but can be modified to pursue novel protein targets for drug discovery in other diseases as well. Chapter 1 of this dissertation provides an explanation of the scientific background and previous research that has motivated the current work. Chapter 2 presents the first high-throughput screening (HTS) platform that detects structural changes in death receptor 5 (DR5), a membrane protein which regulates apoptosis. In this chapter we have identified small-molecules which modulate DR5 signaling and sensitize TRAIL-resistant cancer cells to TRAIL-induced apoptosis. In chapter 3, the same HTS platform is used to discover novel inhibitors of DR5-mediated TRAIL-induced apoptosis for therapeutic intervention of NAFLD and Alzheimer’s Disease. Chapter 4 provides a summary of various projects which involved research on alpha-synuclein (α-syn), the primary protein involved in Parkinson’s Disease. In chapter 4.2, yeast display is implemented as a screening platform to identify inhibitors of α-syn uptake via targeting neurexin-1β. Chapter 4.3 provides an in-depth protocol on the purification of α-syn for laboratory studies. Chapter 4.4 summarizes an attempt to create a cell-free FRET-based biosensor to target the pathological aggregation of α-syn. Chapter 4.5 details a protocol for producing dopamine-modified α-syn oligomers for laboratory studies on dopamine-induced α-syn toxicity. Finally, chapter 4.6 identifies threonine 75 in α-syn as a critical amino acid for fibrillization. This work has overall contributed to the field of drug discovery by providing novel high-throughput screening tools to study DR5 and other protein-protein interactions, and has further elucidated important aspects of α-syn aggregation to be targeted in Parkinson’s Disease.