Induced Tauopathy in a Novel 3D-Culture Model Mediates Neurodegenerative Processes: A Real-Time Study on Biochips
Figure 2
Analysis of cellular alterations after okadaic acid induced hyperphosphorylation in wildtype and mutated tau expressing SH-SY5Y spheroids.
A. Western blot analysis of five days cultivated WT, P301L and K280q tau expressing SH-SY5Y spheroids after incubation with okadaic acid (OA) for 24 hours. Hyperphosphorylation was proven by determination of the pathological relevant tau epitopes T212 (p-tauT212), S262 (p-tauS262) and S422 (p-tauS422). Cellular degradation was analyzed by the caspase-3 cleavage products tauD421 (cl-tau) and cleaved-PARP (cl-PARP) as well as neurofilament-L degradation (NF-L). B. The phosphorylation of the three tau epitopes was quantified relative to total tau, NF-L expression and cleaved protein amounts were quantified relative to β-tubulin (β-tub) C. Significant changes between the tau variants were analyzed to proof the tau pathology state in the tau mutants. (n = 3, *p<0.05, **p<0.01, ***p<0.001). D. For analysis of tauopathy-related alterations in cellular localization and aggregation state of the pathological tau conformation MC-1 (red) and E. tau phosphorylated at S262 (red), five days cultivated WT and K280q spheroids were incubated with 10 nM OA for 24 hours. Immunocytochemical staining of untreated WT, P301L and K280q tau expressing spheroids showed the sporadic accumulation (arrows) and aggregation (arrowheads) of pathological microtubule-associated tau protein. Incubation with OA led to a spread accumulation of aggregated MC-1 and p-tauS262 tau (arrowheads). Fibrous structures indicate the existence of tau fibrils. (bar = 10 µm, nuclear stain DAPI blue).