Myricetin slows liquid–liquid phase separation of Tau and activates ATG5-dependent autophagy to suppress Tau toxicity

Zhi-Xian Chen,Yi Liang,Wang Chen,Bin Dai,Xiao-Ling Liu,Na Zhang,Jie Chen,Tao Zhong,Li-Qiang Wang

doi:10.1016/j.jbc.2021.101222

Zhi-Xian Chen, Yi Liang + Show 7 more

Open Access

https://doi.org/10.1016/j.jbc.2021.101222

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Abstract

Intraneuronal neurofibrillary tangles composed of Tau aggregates have been widely accepted as an important pathological hallmark of Alzheimer's disease. A current therapeutic avenue for treating Alzheimer's disease is aimed at inhibiting Tau accumulation with small molecules such as natural flavonoids. Liquid–liquid phase separation (LLPS) of Tau can lead to its aggregation, and Tau aggregates can then be degraded by autophagy. However, it is unclear whether natural flavonoids modulate the formation of phase-separated Tau droplets or promote autophagy and Tau clearance. Here, using confocal microscopy and fluorescence recovery after photobleaching assays, we report that a natural antioxidant flavonoid compound myricetin slows LLPS of full-length human Tau, shifting the equilibrium phase boundary to a higher protein concentration. This natural flavonoid also significantly inhibits pathological phosphorylation and abnormal aggregation of Tau in neuronal cells and blocks mitochondrial damage and apoptosis induced by Tau aggregation. Importantly, using coimmunoprecipitation and Western blotting, we show that treatment of cells with myricetin stabilizes the interaction between Tau and autophagy-related protein 5 (ATG5) to promote clearance of phosphorylated Tau to indirectly limit its aggregation. Consistently, this natural flavonoid inhibits mTOR pathway, activates ATG5-dependent Tau autophagy, and almost completely suppresses Tau toxicity in neuronal cells. Collectively, these results demonstrate how LLPS and abnormal aggregation of Tau are inhibited by natural flavonoids, bridging the gap between Tau LLPS and aggregation in neuronal cells, and also establish that myricetin could act as an ATG5-dependent autophagic activator to ameliorate the pathogenesis of Alzheimer's disease.

Highlights

The major physiological function of Tau, a microtubuleassociated protein, is the stabilization of neuronal microtubules through the microtubule-binding region of Tau protein [1,2,3,4,5,6]
5 (Fig. 1, A and E), 10 (Fig. 1, B and F), 15 (Fig. 1, C, G, I, and K) or 20 (Fig. 1, D, H, J, and L) μM full-length Tau protein was labeled by 5(6)-carboxy-tetramethylrhodamine N-succinimidyl ester (TAMRA, red fluorescence) and incubated with 10 mM HEPES buffer containing 10% (w/v) polyethylene glycol (PEG) 4000 and 2 mM β-mercaptoethanol or incubated with the same buffer further containing 10 μM myricetin at 25 C to induce liquid–liquid phase separation (LLPS) for 5 min
We show that myricetin slows LLPS of full-length Tau protein and significantly inhibits the subsequent Tau aggregation

Summary

Introduction

The major physiological function of Tau, a microtubuleassociated protein, is the stabilization of neuronal microtubules through the microtubule-binding region of Tau protein [1,2,3,4,5,6]. SH-SY5Y cells stably expressing full-length human Tau formed phosphorylated Tau aggregates when incubated with 10 μM Congo red for 2 days and treated with 0, 5, or 10 μM myricetin for 2 days (Fig. 4, A and C, and Fig. S3).

Results

Conclusion