Metformin activates chaperone-mediated autophagy and improves disease pathologies in an Alzheimer disease mouse model

Chaperone-mediated autophagy (CMA) is a lysosome-dependent selective degradation pathway implicated in the pathogenesis of cancer and neurodegenerative diseases. However, the mechanisms that regulate CMA are not fully understood. Here, using unbiased drug screening approaches, we discover Metformin, a drug that is commonly the first medication prescribed for type 2 diabetes, can induce CMA. We delineate the mechanism of CMA induction by Metformin to be via activation of TAK1-IKKα/β signaling that leads to phosphorylation of Ser85 of the key mediator of CMA, Hsc70, and its activation. Notably, we find that amyloid-beta precursor protein (APP) is a CMA substrate and that it binds to Hsc70 in an IKKα/β-dependent manner. The inhibition of CMA-mediated degradation of APP enhances its cytotoxicity. Importantly, we find that in the APP/PS1 mouse model of Alzheimer’s disease (AD), activation of CMA by Hsc70 overexpression or Metformin potently reduces the accumulated brain Aβ plaque levels and reverses the molecular and behavioral AD phenotypes. Our study elucidates a novel mechanism of CMA regulation via Metformin-TAK1-IKKα/β-Hsc70 signaling and suggests Metformin as a new activator of CMA for diseases, such as AD, where such therapeutic intervention could be beneficial. Supplementary Information The online version contains supplementary material available at 10.1007/s13238-021-00858-3.

(C, D) H4 cells were transfected with indicated siRNA for 12 h, treated with or without 20 μM Metformin for another 48 h, cell lysates were immunoblotted with indicated antibodies.
(E) 293THK cells were pretreated with or without 1 μg/mL DOX, transfected with siRNA of Hsc70 for 12 h, treated with or without 20 μM Metformin for another 48 h, fluorescence of HK2-GFP was analyzed by flow cytometry.
(F) 293THK cells were treated as shown in (E). Cell lysates were immunoblotted with indicate antibodies.
(G, H) HEK293T cells were transfected with indicated plasmids for 24 h, treated with or without 20 μM Metformin for 12 h, the interaction of HK2, PKM2 with Hsc70 and Lamp2a were analyzed by immunoprecipitation. (F) HEK293T cells were transfected with Flag or Hsc70 WT-Flag for 24 h, treated with or without 20 mM Metformin for another 6 h, the interaction of IKKα and Hsc70 was detected by immunoprecipitation.
(G) 293THK cells were pretreated with or without 1 μg/mL DOX and transfected with siRNA of IKKα for 48 h, treated with or without 20 mM Metformin for another 12 h, fluorescence of HK2-GFP was analyzed by flow cytometry (data represents Mean ± SD; ****p < 0.0001, one-way ANOVA).
(H) 293THK cells were treated as in (G). Cell lysates were immunoblotted with indicated antibodies.
(I) H4 cells were transfected with siRNA of IKKα for 12 h, treated with or without 20 μΜ Metformin for another 48 h, cell lysates were immunoblotted with indicated antibodies.
(J) HEK293T cells were transfected with IKKα-Myc for 24 h, treated with or without 20 mM Metformin for 6 h and 8 h, and cell lysates were immunoblotted with indicated antibodies.

Supplementary Fig. 5 Metformin induces phosphorylation of Hsc70 at Ser85 via IKKα/β activation in a TAK1-dependent manner.
(A) 293THK cells were pretreated with or without 1 μg/mL DOX, and transfected with indicated siRNA for 48 h, treated with or without 20 mM Metformin for another 12 h, cell lysates were immunoblotted with indicated antibodies.
(B) 293THK cells were pretreated with or without 1 μg/mL DOX and transfected with siRNA of IKKβ for 12 h, treated with or without 20 μM Metformin for another 48 h, fluorescence of HK2-GFP was analyzed by flow cytometry (data represents Mean ± SD; ****p < 0.0001, one-way ANOVA).
(C) 293THK cell were treated as in (B). Cell lysates were immunoblotted with indicated antibodies.
(D) 293THK cells were pretreated with or without 1 μg/mL DOX, treated with 20 μM Metformin for 36 h, and with or without 5 μM TPCA1 for another 12 h, fluorescence of HK2-GFP was analyzed by flow cytometry.
(E) 293THK cells were treated as shown in (D). Cell lysates were immunoblotted with indicated antibodies.
(F) H4 cells were transfected with siRNA of IKKβ for 12 h, treated with or without 20 μΜ Metformin for another 48 h, cell lysates were immunoblotted with indicated antibodies.
(G) HEK293T cells were transfected with IKKβ for 24 h, treated with or without 20 mM Metformin for 6 h and 8 h. Cell lysates were immunoblotted with indicated antibodies.
(H) 293THK cells were pretreated with or without 1 μg/mL DOX and transfected with siRNA of TAK1 for 12 h, treated with or without 20 μM Metformin for 48 h, cell lysates were immunoblotted with indicated antibodies.