cultures [11,12]. Especially in the field of neurodegenerative diseases, neuronal differentiation, neurite formation and spatial orientation in tissue-like cultures are crucial for the development of an AD-like pathology [13]. To recapitulate the AD-related tau pathology, we generated three SH-SY5Y cell lines that stably overexpress N-terminal EGFP-fused human tau (0N4R) variants: the wildtype (WT), the single mutated tau variant P301L and a novel fourfold mutated variant comprising the single point mutations DK280, P301L, V337M, R406W. These mutations have been identified in frontotemporal dementia and parkinson- ism-linked to chromosome 17 (FTDP-17) related taupathology and are widely used to recapitulate AD-like tau pathology in in vivo models [14]. In contrast to methods like lipofection, calcium phosphate precipitation or even adenoviral transduction the lentiviral transduction of the WT and mutated 4R tau variants ensures a homogenous and moderate stable expression of transgenic tau with a 10–15 fold increased expression level in comparison to native SH-SY5Y cells and an expression level comparable to primary neuronal in vitro cultures [15,16]. To achieve a reproducible tau-specific pathology in an appropriate time scale of several days, we further used the selective protein phosphatase 2A inhibitor okadaic acid (OA) that is commonly used to induce tau hyperphosphorylation in vitro [8,17] and in vivo [18]. Despite the advantages of more complex 3D culture models, there are only limited possibilities for sensitive quantifications of cellular alterations. Especially in the context of label-free real time monitoring for the establishment of standardized screening assays, we developed an electrical impedance spectroscopy-based (EIS) read-out system [8,19] that, in combination with a unique microcavity array [16,20], results in an outstanding tool for the investigations of pathologic processes and moreover, for screening of potential APIs. For demonstrating the performance of our system, we used reference compounds that have previously shown therapeutic benefits in mice models [21,22]. In detail, we investigated the efficacy of a specific (AR-A014418) and a non- specific GSK-3b inhibitor (hymenialdisine) and the HSP-90 inhibitor 17-allylamino-17-demothoxygeldanamycin (17-AAG), which had proven to prevent tau hyperphosphorylation [23,24,25], as well as the tau aggregation inhibitor methylene blue that was evaluated in a phase II trial [26]. Results 3D Cultivation of SH-SY5Y Cells Leads to Increased Neuronal Differentiation With the objective to establish a 3D in vitro model for the label- free monitoring of tauopathy-related degeneration of neuronal cells, especially neuritic processes, that is highly reproducible and feasible for high-content screening systems, we generated SH- SY5Y cell lines stably expressing an N-terminal fused EGFP tau protein by lentiviral transduction. In detail, we established three SH-SY5Y cell lines overexpressing human 0N4R tau variants: wildtype (WT), the common used single point mutation P301L and moreover, a novel fourfold mutation variant K280q comprising the DK280, P301L, V337M, R406W mutations. The poly-mutation was generated with the aim to strengthen the tauopathy. Since SH-SY5Y cells are derived from a neuroblastoma, the neuronal differentiation of monolayer cultures is poorly developed and has to be promoted via retinoic acid, staurosporine or neurotrophic factors as NGF or BDNF [13,27]. Since differenti- ation agents were found to affect various signaling pathways, their application would probably influence the tauopathy induction A Novel Tauopathy 3D-Culture Model on a Biochip [28,29]. Thus, we established more organotypic-like spheroid cultures from all three cell lines (Fig. 1A) by using a self-developed gyratory shaker system [30]. The generated spheroids of all cell lines showed a comparable size and shape development over seven days of cultivation (Fig. 1B) and reached an appropriate size for analysis with a diameter preferably greater than 125 mm after four to five days. By visualizing the EGFP-fused wildtype protein in 2D and 3D cultures, it was possible to show a considerably higher number and even elongation of neuronal processes in the 3D cultures (Fig. 1C, Movie S1). To prove the consistent neuronal character of cells within the spheroids, the localization of the neuronal marker NF200 was analyzed by immunocytochemistry. The staining revealed the distribution of neurofilament structures within all cells concentrated in the prolonged neural processes composing a 3D unit but not in the monolayer culture (Fig. 1D). Those findings were supported by Western blot analysis of the neuronal markers synapsin I and b III-tubulin in WT tau expressing cells cultured as monolayers or spheroids. After seven days of cultivation, both neuronal markers showed higher expression in 3D cultures (Fig. 1E). The densitometric analysis revealed an extreme significant increase of synapsin I expression in 3D cultures (10768%) compared to 2D cultures (5968%). Consistently, b III-tubulin expression increased in 3D cultures (9363%) in comparison to 2D cultures (7261%). Together, this impressively shows that using 3D culture systems enhances neuronal differentiation at least for SH-SY5Y cells without the need for artificial differentiation agents. Tau Hyperphosphorylation Leads to Increased Degenerative Processes in Mutated Tau Expressing 3D Cultures To investigate the assumed tau mutation-dependent cellular degeneration, we performed Western blot analysis of tau pathological, axonal and cellular degradation markers. Moreover, we enforced tau pathological processes by incubation with okadaic acid (OA) for 24 hours in a concentration range of 10–100 nM (Fig. 2A). For proofing induced tau hyperphosphorylation, we analyzed the pathological relevant tau epitopes T212, S262 and S422 (Fig. 2B). Furthermore, we compared the named markers between the tau variants for the OA concentration 25 nM (Fig. 2C). Densitometric analysis of three independent experiments revealed that relative phosphorylation of T212 and S262 was comparable in untreated WT (2661% and 59612%), P301L (1764% and 5568%) and K280q (2165% and 54616%) tau expressing cells (Fig. 2B). Furthermore, p-tauT212 WT tau expressing cells showed no significant increase. In contrast, P301L and K280q tau expressing cells revealed a significant rise to 77626% (P301L) and 58613% (K280q) at 100 nM OA. However, significant differences between the tau variants were not visible for 25 nM OA treatment (Fig. 2C). The pathologic effect was increased for the tau epitope S262 where relative phosphor- ylation was very significantly increased to 10764% (P301L) and 91621% (K280q). Again tau variant comparison showed no significant differences at low OA concentrations. A more pronounced phosphorylation impact could be detected for the tau epitope S422, which exhibited a mutation-dependent phos- phorylation level even without OA treatment (WT: 765%; P301L: 1463%; K280q: 2869%) that reached its significant different maximum for P301L and K280q tau at 10 nM OA (64621% and 86618%, respectively) but never for WT tau, even at 100 nM OA (38615%). Tau variant differences could be determined for the moderate OA concentration 25 nM, the S422 phosphorylation of tau mutants were found to be significant (P301L) and very significant different (K280q) from the WT tau. PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e49150