The neuropathological process in Alzheimer's disease (AD) is characterized by both intra and extracellular Aβ42 aggregates. The neuropathological process of AD is complex and the exact cause of Aβ aggregation leading towards neuronal death is yet unknown. Several events are implicated towards the development of AD including changes within the proteome. With more than 30 million people currently affected with AD, there is still no cure for AD. In this project we seek to identify differential protein profiles by undertaking a comparative analysis of the intracellular and extracellular effects of Aβ on the human proteome using two cellular neuronal models: MC65 and SHSY5Y cells, to understand the biochemical pathology underlying AD. We also initiated a compound screening approach which not only identified several small molecules and peptides inhibiting the Aβ cytotoxicity, but also identified several known compounds from the LOPAC library acting as potential inhibitors of intra and extracellular Aβ42 cytotoxicity, thus highlighting the importance of drug repositioning to identify novel compounds in the therapeutic regime of AD which could be categorized as Aβ toxicity inhibitors. A comparative qualitative proteomics approach was undertaken using OFFGEL fractionation. The MS data was analysed through GO, biological pathway and protein interaction analysis using various databases such as UniProtKB, DAVID v6.7, KEGG and String 9.0 for the SHSY5Y cells treated with extracellular Aβ42 and MC65 cells which conditionally express intracellular C99, that is further cleaved to intracellular Aβ. This was followed by validation of 8 proteins by in-cell Western assay (ICW) undertaken using the LI-COR Infrared Imaging System for the cell lysates of control and Aβ42 treated SH-SY5Y as well as Aβ induced MC65 cells. We have also screened a library of 1280 LOPAC compounds on both the cell lines and 9 other compounds previously known as Aβ toxicity inhibitors on MC65 cells. The lead compounds were further characterized using MTT, LDH, ThT and ICW assays. The proteomics methodology undertaken through this project identified several novel proteins specific to intracellular and extracellular Aβ aggregation. The GO, biological pathway analysis and the functional interaction study helped to identify proteins associated from the proteasome pathway to be affected as an effect of Aβ aggregation for both the cells exposed with intra and extracellular Aβ aggregation. The compound screening study also identified several compounds as inhibitors of Aβ cytotoxicity. A-77636, a D1 dopamine receptor agonist was identified as a lead compound to reduce the extracellular Aβ42 cytotoxicity at nM concentration. Moreover, 1,3-Diethyl-8-phenylxanthine and Arecaidine propargyl ester hydrobromide also proved successful in attenuating the extracellular Aβ42 cytotoxicity. Apart from this; SEN1000, SEN304 and Scylloinositol were able to completely attenuate the intracellular Aβ cytotoxicity, whereas two other compounds, 1,3-Dipropyl-8-p-sulfophenylxanthine and 3-Bromo-7-nitroindazole from the LOPAC library proved effective in acting as partial inhibitors of intracellular Aβ aggregation induced cytotoxicity. The ADME profile for most of these compounds is acceptable, therefore these can be considered as therapeutic leads for AD in the future.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:764270 |
| Date | January 2013 |
| Creators | Modak, Swananda Rajan |
| Contributors | Doig, Andrew |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/effects-of-a42-on-the-human-proteome-and-compound-library-screening-using-cellular-models-of-alzheimers-disease(4cca38b1-fd3f-4ef1-bde1-e16cf227ef68).html |
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