Investigation of the Protective Effect of Specific Inhibitors of Alpha-Synuclein Aggregation
Author
Svanborg, Maria
Term
4. term
Education
Publication year
2011
Pages
104
Abstract
Dette speciale undersøger den beskyttende effekt af specifikke hæmmere af α-synuclein-aggregering, som er central i patogenesen ved Parkinsons sygdom. Oligodendrogliale rottecellelinjer (OLN-AS med α-syn og tau40 samt OLN-t40 med tau40) blev transiente transfekteret med p25α (TPPP), der in vitro kan stimulere α-syn-aggregering, og celleviabilitet blev vurderet med et XTT-assay efter behandling med de udvalgte inhibitorer ASI1D, EGCG og baicalein. Western blotting anvendt til at verificere p25α-udtrykkelse viste tydelige signaler for α-syn og α-tubulin, men ikke for p25α, hvilket peger på lav transfektionseffektivitet og begrænser fortolkningen af inhibitorernes effekter. Under disse betingelser sås ingen markante forskelle i viabilitet efter ASI1D eller EGCG givet en time før transfektion, om end 2 μM ASI1D indikerede en mulig beskyttende effekt i forhold til den ubehandlede negative kontrol, mens baicalein viste cytotoksicitet (relativ viabilitet 45 % ± 25 %). Derudover blev 56 CureND-inhibitorforbindelser screenet for basal cytotoksicitet i OLN-AS, og en ny cellelinje, OLN-AS7 uden tau40, blev taget i brug (fordoblingstid 18,7 timer). For at muliggøre fremtidige MS-baserede (SILAC) undersøgelser af mitokondrielle subproteomer ved inducerbar p25α-udtrykkelse blev et inducerbart ekspressionssystem igangsat, inklusive konstruktion og verifikation af p25α-pEGSH og p25α-GFP-pEGSH samt fastlæggelse af selektionsbetingelser (LD100: hygromycin 200 μg/mL efter 11 dage; geneticin 600 μg/mL efter 15 dage).
This thesis investigates the protective effect of specific inhibitors of α‑synuclein aggregation, a process central to Parkinson’s disease pathogenesis. Oligodendroglial rat cell models (OLN-AS expressing α‑syn and tau40, and OLN‑t40 expressing tau40) were transiently transfected with p25α (TPPP), which can stimulate α‑syn aggregation in vitro, and cell viability was assessed using an XTT assay after treatment with the inhibitors ASI1D, EGCG, and baicalein. Western blotting used to verify p25α expression showed clear signals for α‑syn and α‑tubulin but not for p25α, indicating low transfection efficiency and limiting interpretation of inhibitor effects. Under these conditions, no substantial viability differences were observed after ASI1D or EGCG administered one hour before transfection, although 2 μM ASI1D suggested a possible protective effect relative to the untreated negative control, while baicalein appeared cytotoxic (relative viability 45% ± 25%). Additionally, 56 CureND inhibitor compounds were screened for baseline cytotoxicity in OLN‑AS, and a new cell line, OLN‑AS7 lacking tau40, was introduced (doubling time 18.7 hours). To enable future MS‑based (SILAC) studies of mitochondrial subproteomes with inducible p25α expression, an inducible expression system was initiated, including construction and verification of p25α‑pEGSH and p25α‑GFP‑pEGSH, and determination of selection conditions (LD100: hygromycin 200 μg/mL at 11 days; geneticin 600 μg/mL at 15 days).
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