Endogenous tau released from humanReNCell VMcultures by neuronal activity is phosphorylated at multiple sites

Abstract

Tau is an intracellular protein but also known to be released into the extracellular fluid. Tau release mechanisms have drawn intense attention as these are known to play a key role in Alzheimer’s disease (AD) pathology. However, tau can also be released under physiological conditions although its physiological function and release mechanisms have been poorly characterized, especially in human neuronal cells.We investigated endogenous tau release inReNCellVM, a human neuroprogenitor cell line, under physiological conditions and found that tau is spontaneously released from cells. To study activity-dependent release of endogenous tau, humanReNCell VMculture was stimulated by 100μM AMPA or 50mM KCl for one-hour, tau was actively released to the culture medium. The released tau was highly phosphorylated at nine phosphorylation sites (pSites) detected by phospho-specific tau antibodies including AT270 (T175/T181), AT8 (S202/T205), AT100 (T212/S214), AT180 (T231), and PHF-1 (S396/S404), showing that these pSites are important for activity-dependent tau release from humanReNCellVM. Intracellular tau showed various phosphorylation status across these sites, with AT270 and PHF-1 highly phosphorylated while AT8 and AT180 were minimally phosphorylated, suggesting that AT8 and AT180 pSites exhibit a propensity for secretion rather than being retained intracellularly. This activity-dependent tau release was significantly decreased by inhibition of GSK-3β, demonstrating that GSK3β-dependent phosphorylation of tau plays an important role in its release by neuronal activity.In this study, we showed thatReNCellVM serves as a valuable model for studying endogenous physiological tau release. Further,ReNCellmodel can be also used to study pathological release of human tau that will contribute to our understanding of the progression of AD and related dementias.HighlightsActivity-dependent release of endogenous human tau from human ReNCell VM cultures occurs under physiological conditions.Released human tau is phosphorylated at nine sites (pSites) in the proline-rich domain and the C-terminal domain detected by AT270 (T175/T181), AT8 (S202/T205), AT100 (T212/S214), AT180 (T231), and PHF-1 (S396/S404) tau antibodies, strongly suggesting that these pSites are important for activity-dependent tau release from human ReNCell VM.In contrast, intracellular human tau proteins have different phosphorylation status among these nine pSites: AT270 and PHF-1 pSites are highly phosphorylated, but AT8 and AT180 are weakly phosphorylated, suggesting AT8 and AT180 pSites are release-sensitive phosphorylation motifs.Activity-dependent release of endogenous human tau is decreased by a tau kinase GSK-3β inhibitor SB 216763, indicating that GSK-3β-dependent phosphorylation plays an important role in activity-dependent tau release.The humanReNCellculture is an excellent model system to study mechanisms underlying physiological release of endogenous tau.