Figure 1. 3D cultivation of tau expressing SH-SY5Y cells leads to enhanced neuronal differentiation. A. Stably EGFP-tau fusion protein expressing SH-SY5Y cells were cultivated on a gyratory shaker in order to generate spheroids within the first five days in vitro (DIV) (bar = 100 mm). B. Statistical analysis of spheroid diameter revealed comparable size development over time for wildtype (WT), single- mutated (P301L) and poly-mutated (K280q) tau expressing SH-SY5Y cells (mean 6 s.d.). C. Live cell imaging of the EGFP-fused wildtype tau A Novel Tauopathy 3D-Culture Model on a Biochip (green) and D. immunocytochemical staining of the neuronal marker NF200 (red) ensured the overall existence of matured neural cells and revealed extended neuritic processes (arrows) in five days cultivated spheroids (3D) in comparison to monolayer cultures (2D) (bar = 20 mm, nuclear stain DAPI blue). E. The expression of the neuronal markers synapsin I (syn I)and b III-tubulin (bIII-tub) in wildtype tau expressing 2D and 3D cultures at culture day seven were analyzed via Western blot and quantified relative to the housekeeping protein b-tubulin.(n $3, **p,0.01, ***p,0.001). doi:10.1371/journal.pone.0049150.g001 Interestingly, analysis of the axonal marker protein neurofilament- L (NF-L) showed a slight non-significant increase of expression at lower OA concentrations. Higher concentrations of OA induced a progressive and significant reduction of NF-L expression to 32617% (WT), 1264% (P301L) and 2065% (K280q) implying an advanced neurite and cellular degeneration. More strikingly, significant differences between the tau mutants and the WT at 25 nM could be detected. Next, we analyzed the amount of caspase 3-cleaved tau. The C-terminal truncation of tau (Asp421) at 25 nM OA was found to be doubled in WT tau expressing spheroids, whereas OA treatment in P301L and K280q tau expressing spheroids led to 15 and 12 times higher amounts compared to the control. In contrast, the analysis of the caspase 3-cleaved amount of PARP (Asp214) showed a similar progression for all three cell lines. However, treatment with 25 nM OA induced an extreme significant increase of cleaved PARP amounts to 4668% in WT, 71611% in P301L and 8569% in K280q 3D cultures. At this concentration, significant differences between the WT tau cell line and the mutants were detectable for both cl-tau and cl- PARP amounts. Interestingly, 10 nM OA was in no case sufficient to significantly change the amounts of these apoptosis markers. However, immunocytochemical stainings showed that the amount of aggregated tau was increased after 24 hour incubation with 10 nM OA (Fig. 2D2E). Especially, the conformational-dependent MC-1 antibody that recognizes a pathological tau conformation [31,32] revealed low molecular as well as high molecular aggregated tau. While WT tau spheroids exhibited low molecular weight tau accumulation (arrows) especially after treatment with OA, the local aggregation of pathological folded tau was present even in untreated P301L and K280q cells. The incubation with 10 nM OA resulted in high molecular weight tau aggregates in individual cells in a mutation number-dependent pattern (arrowheads). A similar pattern but with increased progression was detected for stained p-tauS262 (Fig. 2E). Whereas high molecular weight aggregates (arrows) and low molecular weight aggregates of p-tauS262 positive tau (arrowheads) were visible in individual untreated WT tau cells, the number of affected cells and hyperpho- sphorylated tau deposits was greater for cells with increased mutation number and OA treatment. In the highly pathological K280q tau expressing cells, the application of 10 nM OA for 24 hours was sufficient to induce the formation of p-tauS262 deposits in fibrous structures. Taken together the aggregation patterns of pathological tau, the changed phosphorylation levels of pathological relevant tau epitopes and amounts of NF-L as well as the missing incidence of apoptosis markers up to 25 nM OA proof a selective tauopathy induction in a mutation-dependent manner. However, at higher OA concentrations cytotoxic effects determine the cell fate. PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e49150