Naslov
Okadaic acid treated SH-SY5Y neuroblastomacell line as a model to study tau protein phosphorylation and oligomerization

Autori
Boban, Mirta ; Miškić, Terezija ; Babić Leko, Mirjana ; Šimić, Goran

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
6. Croatian Neuroscience Congress Book of Abstracts / - , 2017, 89-89

Skup
6. Croatian Neuroscience Congress

Mjesto i datum
Osijek, Hrvatska, 16.09.2017. - 18.09.2017

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
Alzheimer's disease ; neurodegeneration ; cell culture ; SH-SY5Y ; okadaic acid ; oligomerization ; phosphorylation ; protein phosphatase ; tau protein ; immunoblot ; cycloheximide

Sažetak
A key feature in the early stages of Alzheimer’s disease is abnormal phosphorylation of tau, a microtubule associated protein. Tau hyperphosphorylation results from the activity of several protein kinases and down regulation of phosphatase PP2A. To study processes involving phosphorylated tau, cultured cells can be treated with okadaic acid (OA), a phosphatase inhibitor that primarily targets PP2A. SH-SY5Y cells express several splicing isoforms of tau and are useful as a cell culture model for investigating pathways important for neuronal tissues due to their neuron-like characteristics. To induce accumulation of hyperphosphorylated tau, in vitro cultured SH-SY5Y neuroblastoma cells were treated with 100 nM OA for variable periods of time. Cells were harvested and protein lysates prepared. Tau protein levels and phosphorylation status at specific amino acid residues were analyzed by immunoblot(western blot) using antibodies against N-terminal part of tau(CP27) or middle part of tau(Tau5) and phospho-tau specific antibodies(CP13 and anti-tau-pSer396). Levels of glyceraldehyde-3- phosphate dehydrogenase(GAPDH) were used as a loading control. In the experiment that required inhibition of protein synthesis, we treated cells with 100 mM cycloheximide for 15 min prior to adding OA. We observed that OA-treated SH-SY5Y cells express tau protein of the apparent molecular weight around 100 kDa, in addition to the well-described 45-65 kDa tau isoforms. High molecular weight tau was detected by western blot using antibodies against tau phosphorylated at amino acid residues Ser202 and Ser396, as well as by the CP27 antibody that recognizes N-terminal part of tau. Cells that have been pretreated with cycloheximide, a general inhibitor of protein synthesis, express 100 kDa tau protein upon treatment with OA, showing that high molecular weight tau is not newly synthesized in response to OA treatment, and may thus represent tau oligomer. Biochemical characterization showed that 100 kDa tau is stable in the presence of strong denaturing and chaotropic agents urea and guanidine. 100 kDa tau did not dissociate in the solution containing beta-mercaptoethanol, indicating that the potential oligomer does not require disulfide bonds. Our results show that treatment of SH-SY5Y cells with OA induces formation of high molecular weight tau protein that may represent a large splicing isoform of tau or tau oligomer. In support of the latter possibility, a previous study using fluorescently labeled tau transfected into HEK293 cells indicated that tau oligomerizes upon cell treatment with OA (Taket al., PLOS One 2013, 8:12). We biochemically characterized 100 kDa tau and excluded a possibility that this protein is newly synthesized as a response to OA treatment. Our findings contribute to characterization of SH-SY5Y cell culture treated with OA as a model for studying phospho-tau related processes.

Izvorni jezik
Engleski

Znanstvena područja
Biologija, Temeljne medicinske znanosti, Kliničke medicinske znanosti

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