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

Abstract

Chaperone-mediated autophagy (CMA) is a lysosomedependent 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.

Key words： chaperone-mediated autophagy; Metformin; TAK1; IKKα/β; Hsc70; APP; Alzheimer’s disease

References

1 Agarraberes FA, Dice JF(2001) A molecular chaperone complex at the lysosomal membrane is required for protein translocation. J Cell Sci 114:2491–2499

2 Agarraberes FA, Terlecky SR, Dice JF(1997) An intralysosomal hsp70 is required for a selective pathway of lysosomal protein degradation. J Cell Biol 137:825–834

3 Barzilai N, Crandall JP, Kritchevsky SB, Espeland MA(2016) Metformin as a tool to target aging. Cell Metab 23:1060–1065

4 Bhat A, Sebastiani G, Bhat M(2015) Systematic review: Preventive and therapeutic applications of metformin in liver disease. World J Hepatol 7:1652–1659

5 Breining P (2018) Metformin targets brown adipose tissue in vivo and reduces oxygen consumption in vitro. Diabetes Obes Metab 20:2264–2273

6 Campbell JM (2018) Metformin use associated with reduced risk of dementia in patients with diabetes: A systematic review and meta-analysis. J Alzheimers Dis 65:1225–1236

7 Chen F(2016) Antidiabetic drugs restore abnormal transport of amyloid-beta across the blood-brain barrier and memory impairment in db/db mice. Neuropharmacology 101:123–136

8 Chiang HL, Terlecky SR, Plant CP, Dice JF(1989) A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins. Science 246:382–385

9 Chromy BA(2003) Self-assembly of Abeta(1–42) into globular neurotoxins. Biochemistry 42:12749–12760

10 Comb WC, Hutti JE, Cogswell P, Cantley LC, Baldwin AS(2012) p85alpha SH2 domain phosphorylation by IKK promotes feedback inhibition of PI3K and Akt in response to cellular starvation. Mol Cell 45:719–730

11 Cox J, Mann M(2008) MaxQuant enables high peptide identification rates, individualized ppb-range mass accuracies and proteomewide protein quantification. Nat Biotechnol 26:1367–1372

12 Criollo A(2010) The IKK complex contributes to the induction of autophagy. EMBO J 29:619–631

13 Cuervo AM, Dice JF(1996) A receptor for the selective uptake and degradation of proteins by lysosomes. Science 273:501–503

14 Cuervo AM, Wong E(2014) Chaperone-mediated autophagy: roles in disease and aging. Cell Res 24:92–104

15 Diabetes Prevention Program Research, G. Long-term safety, tolerability, and weight loss associated with metformin in the Diabetes Prevention Program Outcomes Study. Diabetes Care 35, 731–737 (2012).

16 Dice JF(1990) Peptide sequences that target cytosolic proteins for lysosomal proteolysis. Trends Biochem Sci 15:305–309

17 Dice JF, Chiang HL, Spencer EP, Backer JM(1986) Regulation of catabolism of microinjected ribonuclease A. Identification of residues 7–11 as the essential pentapeptide. J Biol Chem 261:6853–6859

18 Ea CK, Deng L, Xia ZP, Pineda G, Chen ZJ(2006) Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol Cell 22:245–257

19 Evans JM, Donnelly LA, Emslie-Smith AM, Alessi DR, Morris AD(2005) Metformin and reduced risk of cancer in diabetic patients. BMJ 330:1304–1305

20 Fezoui Y (2000) A de novo designed helix-turn-helix peptide forms nontoxic amyloid fibrils. Nat Struct Biol 7:1095–1099

21 Gandini S(2014) Metformin and cancer risk and mortality: a systematic review and meta-analysis taking into account biases and confounders. Cancer Prev Res (phila) 7:867–885

22 Gough NR, Fambrough DM(1997) Different steady state subcellular distributions of the three splice variants of lysosome-associated membrane protein LAMP-2 are determined largely by the COOHterminal amino acid residue. J Cell Biol 137:1161–1169

23 Graham GG(2011) Clinical pharmacokinetics of metformin. Clin Pharmacokinet 50:81–98

24 Groftehauge MK, Hajizadeh NR, Swann MJ, Pohl E(2015) Proteinligand interactions investigated by thermal shift assays (TSA) and dual polarization interferometry (DPI). Acta Crystallogr D Biol Crystallogr 71:36–44

25 Guo Q, Wang Z, Li H, Wiese M, Zheng H(2012) APP physiological and pathophysiological functions: insights from animal models. Cell Res 22:78–89

26 Hardie DG, Ross FA, Hawley SA(2012) AMP-activated protein kinase: a target for drugs both ancient and modern. Chem Biol 19:1222–1236

27 Hettich MM (2014) The anti-diabetic drug metformin reduces BACE1 protein level by interfering with the MID1 complex. PLoS ONE 9:e102420

28 Kaushik S, Cuervo AM(2015) Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis. Nat Cell Biol 17:759–770

29 Kaushik S, Cuervo AM(2018) The coming of age of chaperonemediated autophagy. Nat Rev Mol Cell Biol 19:365–381

30 Keowkase R (2010) Neuroprotective effects and mechanism of cognitive-enhancing choline analogs JWB 1–84-1 and JAY 2–22-33 in neuronal culture and Caenorhabditis elegans. Mol Neurodegener 5:59

31 Kirchner P(2019) Proteome-wide analysis of chaperonemediated autophagy targeting motifs. PLoS Biol 17:e3000301

32 Kon M (2011) Chaperone-mediated autophagy is required for tumor growth. Sci Transl Med 3:109ra117

33 Labuzek K (2010) Quantification of metformin by the HPLC method in brain regions, cerebrospinal fluid and plasma of rats treated with lipopolysaccharide. Pharmacol Rep 62:956–965

34 Lamanna C, Monami M, Marchionni N, Mannucci E(2011) Effect of metformin on cardiovascular events and mortality: a metaanalysis of randomized clinical trials. Diabetes Obes Metab 13:221–228

35 Lane CA, Hardy J, Schott JM(2018) Alzheimer’s disease. Eur J Neurol 25:59–70

36 Liu T, Daniels CK, Cao S(2012) Comprehensive review on the HSC70 functions, interactions with related molecules and involvement in clinical diseases and therapeutic potential. Pharmacol Ther 136:354–374

37 Mercurio F(1997) IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation. Science 278:860–866

38 Neven E(2018) Metformin prevents the development of severe chronic kidney disease and its associated mineral and bone disorder. Kidney Int 94:102–113

39 Ng TP (2014) Long-term metformin usage and cognitive function among older adults with diabetes. J Alzheimers Dis 41:61–68

40 Park C, Suh Y, Cuervo AM(2015) Regulated degradation of Chk1 by chaperone-mediated autophagy in response to DNA damage. Nat Commun 6:6823

41 Schneider JL(2015) Loss of hepatic chaperone-mediated autophagy accelerates proteostasis failure in aging. Aging Cell 14:249–264

42 Schneider JL, Suh Y, Cuervo AM(2014) Deficient chaperonemediated autophagy in liver leads to metabolic dysregulation. Cell Metab 20:417–432

43 Scrivo A, Bourdenx M, Pampliega O, Cuervo AM(2018) Selective autophagy as a potential therapeutic target for neurodegenerative disorders. Lancet Neurol 17:802–815

44 Scuderi C, Steardo L, Esposito G(2014) Cannabidiol promotes amyloid precursor protein ubiquitination and reduction of beta amyloid expression in SHSY5YAPP+ cells through PPARgamma involvement. Phytother Res 28:1007–1013

45 Smith DF, Whitesell L, Katsanis E(1998) Molecular chaperones: biology and prospects for pharmacological intervention. Pharmacol Rev 50:493–514

46 Stricher F, Macri C, Ruff M, Muller S(2013) HSPA8/HSC70 chaperone protein: structure, function, and chemical targeting. Autophagy 9:1937–1954

47 Tiwari S, Atluri V, Kaushik A, Yndart A, Nair M(2019) Alzheimer’s disease: pathogenesis, diagnostics, and therapeutics. Int J Nanomedicine 14:5541–5554

48 Tizazu AM(2019) Metformin monotherapy downregulates diabetes-associated inflammatory status and impacts on mortality. Front Physiol 10:572

49 Vakifahmetoglu-Norberg H(2013) Chaperone-mediated autophagy degrades mutant p53. Genes Dev 27:1718–1730

50 Valdor R(2014) Chaperone-mediated autophagy regulates T cell responses through targeted degradation of negative regulators of T cell activation. Nat Immunol 15:1046–1054

51 Vorhees CV, Williams MT(2006) Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 1:848–858

52 Wang C(2001) TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412:346–351

53 Wang Y(2009) Tau fragmentation, aggregation and clearance: the dual role of lysosomal processing. Hum Mol Genet 18:4153–4170

54 Wang X(2017) Modifications and trafficking of APP in the pathogenesis of alzheimer’s disease. Front Mol Neurosci 10:294

55 Wegmann S, Bennett RE, Amaral AS, Hyman BT (2017) Studying tau protein propagation and pathology in the mouse brain using adeno-associated viruses. Methods Cell Biol 141:307–322

56 Wentink ASet al (2020) Molecular dissection of amyloid disaggregation by human HSP70. Nature 587:483–488

57 Wing SS, Chiang HL, Goldberg AL, Dice JF(1991) Proteins containing peptide sequences related to Lys-Phe-Glu-Arg-Gln are selectively depleted in liver and heart, but not skeletal muscle, of fasted rats. Biochem J 275(Pt 1):165–169

58 Woronicz JD, Gao X, Cao Z, Rothe M, Goeddel DV(1997) IkappaB kinase-beta: NF-kappaB activation and complex formation with IkappaB kinase-alpha and NIK. Science 278:866–869

59 Xia HG (2015) Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death. J Cell Biol 210:705–716

60 Xilouri M(2016) Impairment of chaperone-mediated autophagy induces dopaminergic neurodegeneration in rats. Autophagy 12:2230–2247

61 Xu D(2018) TBK1 suppresses RIPK1-driven apoptosis and inflammation during development and in aging. Cell 174:1477–1491 e1419

62 Xu D(2020) Modulating TRADD to restore cellular homeostasis and inhibit apoptosis. Nature 587:133–138

63 Xue Y(2008) GPS 2.0, a tool to predict kinase-specific phosphorylation sites in hierarchy. Mol Cell Proteomics 7:1598–1608

64 Yamagishi N, Ishihara K, Hatayama T(2004) Hsp105alpha suppresses Hsc70 chaperone activity by inhibiting Hsc70 ATPase activity. J Biol Chem 279:41727–41733

65 Yang Y, Wu Y, Zhang S, Song W(2013) High glucose promotes Abeta production by inhibiting APP degradation. PLoS ONE 8: e69824

66 Zhang CS(2016) Metformin activates AMPK through the lysosomal pathway. Cell Metab 24:521–522

67 Zhang J(2021) Inflammation-induced inhibition of chaperonemediated autophagy maintains the immunosuppressive function of murine mesenchymal stromal cells. Cell Mol Immunol 18 (6):1476–1488