Iron-mediated aggregation and toxicity in a novel neuronal cell culture model with inducible alpha-synuclein expression

Parkinson's disease (PD) represents an increasing problem in society. The oligomerization of alpha-synuclein (alpha Syn) is a suggested key event in its pathogenesis, yet the pathological modes of action remain to be fully elucidated. To identify potential disease-modifying therapeutics and to study alpha Syn-mediated toxic mechanisms, we established cell lines with inducible overexpression of different alpha Syn constructs: alpha Syn, alpha Syn coupled to the fluorescence protein Venus (alpha Syn-Venus), and alpha Syn coupled to the N-terminal or C-terminal part of Venus (V1S and SV2, respectively) for a bimolecular fluorescence complementation assay (BiFC). Inducibility was achieved by applying modified GAL4-UAS or Cre-loxP systems and addition of tebufenozide or 4-OH-tamoxifen, respectively. Expression constructs were stably integrated into the host genome of H4 neuroglioma cells by lentiviral transduction. We here demonstrate a detailed investigation of the expression characteristics of inducible H4 cells showing low background expression and high inducibility. We observed increased protein load and aggregation of alpha Syn upon incubation with DMSO and FeCl3 along with an increase in cytotoxicity. In summary, we present a system for the creation of inducibly alpha Syn-overexpressing cell lines holding high potential for the screening for modulators of alpha Syn aggregation and alpha Syn-mediated toxicity.