engleski

Validation of split luciferase reporter system for the detection of human tau protein oligomerization in living yeast cells

One of the main neuropathological hallmarks of Alzheimer’s disease are neurofibrillary tangles (NTFs), large aggregates of microtubule-binding protein tau that form within the affected neurons. The formation of NTFs is preceded by early-stage tau oligomers, however the molecular pathways that initiate tau aggregation are unclear. To better understand early steps in tau pathology, we constructed a tool for studying tau oligomerization in living cells, based on the luminescent reporter NanoBiT, in which protein- protein interaction results in the complementation of the luciferase NanoLuc, and consequently in generation of luminescence that can be measured in living cells. Since molecular pathways of protein aggregation are largely evolutionarily conserved, we selected a simple cell model, yeast Saccharomyces cerevisiae. We separately fused two luciferase subunits to the human tau protein C- terminus. In order to test whether the observed luminescence is a specific result of tau-tau interaction, we constructed negative controls, including cells that carry an empty vector, and cells that express tau fused to only one of the luciferase subunits. Expression of tau constructs was verified using western blot. By measuring luciferase activity in living cells, we showed that cells expressing tau separately fused to two luciferase subunits exhibited an increased luminescence, as compared to controls, indicating the formation of tau oligomers. In order to test whether tau-NanoBiT reporter is able to detect sarkosyl- insoluble tau aggregates, we measured tau-NanoBiT luminescence in pho85Δ, rim1Δ and sod2Δ mutants that were previously reported to have increased levels of sarkosyl-insoluble tau. Our preliminary data showed that the tau- NanoBiT luminescence was elevated in the sod2Δ mutant, while the signal in pho85Δ, and rim1Δ mutants was similar to the wild-type levels. In conclusion, we constructed a luminescent reporter for human tau protein oligomerization in living yeast cells. Our future experiments will address whether tau- NanoBiT reporter correlates with the levels of sarkosyl-insoluble tau aggregates. Research was co-financed by the Scientific Centre of Excellence for Basic, Clinical and Translational Neuroscience (project “Experimental and clinical research of hypoxic-ischemic damage in perinatal and adult brain” ; GA KK01.1.1.01.0007 funded by the European Union through the European Regional Development Fund) and Croatian Science Foundation (grant IP-2014-09-9730 and DOK-01- 2018/ European Social Fund).

Alzheimer's disease, protein aggregation, tau, protein-protein interaction, Saccharomyces cerevisiae

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