dependent manner. Even after treatment with 5 nM OA for only 24 hours significant differences were observed across WT (11364%) and K280q (9863%) tau expressing spheroids. After 72 hours all tau variants showed a relative impedance decrease with a tau mutation number-dependent but not significant effect (WT: 9163%, P301L: 8763%, K280q: 8163%). In contrast, the respective cross section area analysis revealed a mutation number- and time-dependent increase after 48 hours (WT: 10261%, P301L: 10962%, K280q: 11162%). This spheroid cell swelling effect was intensified at 10 nM OA treatment for 24 and 48 hours for the tau mutants. In agreement with this finding, the relative impedance was only decreased in the mutated tau variants with an extreme significant effect (WT: 10864%, P301L: 7863%, K280q: 8663%). The spheroid shape became much more irregular showing blebbed cells. An enhancement of the pathological situation either by prolonged incubation time or a higher OA concentration led to the decrease of both the relative impedance and the cross section area in a significant mutation number- dependent manner. At an OA concentration of 25 nM after a 72 hour incubation period, a strongly declined relative impedance (WT: 5463%, P301L: 1762%, K280q: 661%) could be correlated with a very low relative cross section area (WT: 6761%, P301L: 4863%, K280q: 3062%) indicating proceeding spheroid degradation. Overall, the application of 100 nM OA led to a much faster relative impedance decrease. Hence, the number of significant impedance differences got smaller with prolonged incubation. Also the increase of cross section area after 24 hours was replaced by a decrease for all tau variants reflecting the degeneration of spheroids to small fragments with progressively shrinking cell structure. To correlate the documented cell structure alterations with morphological intercellular degenerative processes, live images of SH-SY5Y cells expressing EGFP-fused tau proteins were taken after a 72 hours treatment with OA (Fig. 3D). While in control spheroids the ordered network of microtubule-associated tau (arrowheads) and the presence of long and defined neuronal processes (arrows) were obvious, the incubation with 25 nM OA led to degradation of the neuronal processes as well as the tau dissociation from the microtubule network, visible in a more homogenously distributed cellular background fluorescence. OA- treated WT tau expressing cells still exhibited small processes, which were almost completely lost in K280q tau expressing spheroids. Furthermore, accumulations of EGFP-tau were visible in 3D cultures, especially in K280q tau expressing cells exposed to 25 nM OA, where neurodegeneration was visible (arrowheads). Altogether, we were able to detect and quantify mutation number- and therewith tau-specific neurodegeneration in a time- and OA concentration-dependent manner with high sensitivity and precision. With the help of apoptosis marker expression analysis and live cell imaging, we were able to distinguish impedimetrically between the retraction of neurites as one essential pathological hallmark of AD and massive cell death that occurred at OA concentrations higher than 25 nM. Thus, comparing apoptosis marker expression and imaging results with the impedimetric analysis, the latter is able to detect pathological alterations much earlier and more specific. Moreover, the increasing and therefore tau-specific differences across the WT tau expressing 3D-cultures and our novel high-pathologic fourfold mutated tau K280q expressing 3D-cultures combined with our automatable impedimetric monitoring system offer a perfect platform for high content compound testing (Fig. 4A). In this context, we identified an OA concentration of 25 nM as most convenient for a suitable pathology monitoring window between A Novel Tauopathy 3D-Culture Model on a Biochip treated WT and K280q cultures that represents the tau-specific effect. Impedance Spectroscopy-based Quantification of Tau- specific Compound Efficacy To demonstrate the capability of our developed system for compound efficacy quantification, we chose four different refer- ence compounds focusing on different tau pathology-related targets and clearing mechanisms, respectively. Therefore, untreat- ed WT and K280q tau expressing spheroids were used that differed in the expression of a non-mutated and mutant tau variant and were characterized by a physiological impedance level (normalized to 100%). To determine the disease recovery potential of the described tau-specific reference compounds, spheroids were treated with 25 nM OA in the absence (defined as pathological impedance level, PL) or presence (defined as pathology recovery) of compounds at different concentrations (10–500 nM) for 24 and 72 hours. Measurements were aimed to identify whether there was a tau-specific pathology recovery (different recovery values for WT and K280q $1), a more common cellular pathology recovery (similar recovery pattern for WT and K280q $1) or even a toxic side effect (declined recovery values for WT and K280q #1) of the compound (Fig. 4A). All values were expressed as x-fold changes compared to the appropriate OA-treated spheroids (25 nM) that represent the pathological level (PL normalized to 1.0). With the aim to identify tau pathology-specific compounds, a higher, most likely significant recovery for K280q is desired. The values for 24 hours and 72 hours treatment (Fig. 4B) as well as relative cross section areas of spheroids were determined (Fig. S1). After 24 hours treatment, only few significant impedance differences between WT and K280q tau spheroids were observed. The relative cross section area analysis also revealed only few significant differences between the two tau variants with WT tau spheroids always showing the higher cross section area. Extreme significant effects were detected after 72 hours incubation. Even small concentrations of the compounds led to three to five times higher recovery values in K280q tau expressing spheroids compared to the WT tau expressing spheroids. Highest tau-specific pathology recovery was observed for 10 nM AR-A014418 (WT: 1.7360.12, K280q: 5.1060.82) and 17-AAG (WT: 1.6760.07, K280q: 5.0760.75) followed by hymenialdisine (WT: 1.5460.09, K280q: 4.5260.61) and methylene blue (WT: 1.3560.07, K280q: 3.2460.53). In contrast, at higher concentrations (500 nM) of hymenialdisine and 17-AAG values below the pathologic level were detected especially in WT tau expressing spheroids (0.5560.08 and 0.7960.08), which may indicate toxic side effects. In contrast, high concentrations of the specific GSK-3 inhibitor AR- A014418 did not cause toxic side effects (WT: 1.0660.06). Moreover, the analysis of the cross section area could provide additional information for a comprehensive view on compound effects (Fig. S1). Treatment with AR-A014418, hymenialdisine and 17-AAG for 72 hours led to an increased cross section area compared to the pure OA-treated samples in both tau variants. In contrast, the incubation with methylene blue provoked recovery values that were always lower than one. However, only few significant cross section area differences between WT and K280q tau spheroids were detected. These findings demonstrate that even small concentrations in the low nanomolar range of the reference compounds led to strongly increased recovery values after prolonged incubation for 72 hours. Furthermore, effects were found to be tau mutation- dependent, since they were much more intensive in the high- pathologic K280q tau expressing 3D cultures than in WT tau PLOS ONE | www.plosone.org 5 November 2012 | Volume 7 | Issue 11 | e49150