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Fig. 3 | Acta Neuropathologica Communications

Fig. 3

From: Tau seeding activity in various regions of down syndrome brain assessed by two novel assays

Fig. 3

Tau seeding activity in the brains of AD and related tauopathies determined by capture and seeded-tau aggregation assays. a Tau is hyperphosphorylated in the brains of AD and related tauopathies. Tau and phospho-tau in the brain extracts of frontal cortices from five AD, five control, three CBD, three PiD, and two PSP cases were analyzed by Western blots developed with 77G7 (pan-tau) and PHF-1 (pS396/404-tau). b–d 3R-tau and 4R-tau were captured by brain extracts from AD and related tauopathies in which tau was hyperphosphorylated. Various amounts of brain extracts were applied onto a NC membrane. The membrane was incubated with cell extracts containing HA-3R-tau151-391 or HA-4R-tau151-391 for capture assay (b). The levels of captured tau were plotted against protein amounts in the brain extracts dotted on the membrane (c). Levels of captured 4R-tau151-391 were plotted against levels of captured 3R-tau151-391. The correlation was analyzed by non-linear regression (d). e AD brain extract seeded tau151-391 aggregation dose-dependently. HEK-293FT cells expressing HA-3R-tau151-391 were treated with various amounts of AD brain extracts for 42 h. RIPA-insoluble and -soluble taus were analyzed by Western blots. The levels of RIPA-insoluble tau (HA immunoreactivity) were plotted against protein amounts of AD brain extracts. f–h Aggregation of 3R-tau and 4R-tau were seeded similarly by the brain extracts of AD and other tauopathies in which tau was hyperphosphorylated. HEK-293FT cells expressing HA-3R-tau151-391 or HA-4R-tau151-391 were treated with brain extracts from five AD, five control, three CBD, three PiD, and two PSP cases for 42 h. RIPA-insoluble and -soluble tau were analyzed by Western blots (f). The levels of RIPA-insoluble tau are presented as mean \(\pm\) SD, with each dot representing an individual brain sample (g). **P < 0.05; ****P < 0.0001. Correlation of RIPA-insoluble 3R-tau151-391 and 4R-tau151-391 seeded by the brain extracts was analyzed by non-linear regression (h). i Tau seeding activity determined by capture assay was consistent with that determined by seeded-tau aggregation assay. Correlation of RIPA-insoluble 4R-tau151-391 seeded and 4R-tau151-391 captured by the brain extracts was analyzed by non-linear regression. Although all tauopathies are characterized by tau pathology of hyperphosphorylated and seeding competent tau, we did not detect it in the tissue pieces used from several cases, suggesting that the pathology is not evenly distributed throughout the frontal cortex in tauopathies

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