Korean J Physiol Pharmacol.  2025 Jan;29(1):57-66. 10.4196/kjpp.24.155.

p66shc deficiency attenuates high glucose-induced autophagy dysfunction in Schwann cells

• Choi SJ
• Vu GH
• Nagar H
• Kim S
• Lee I
• Piao S
• Jeon BH
• Irani K
• Oh SH
• Kim CS

• KMID: 2563500
• DOI: http://doi.org/10.4196/kjpp.24.155

Abstract

Schwann cells are the most abundant cells in the peripheral nervous system, maintaining the development, function and regeneration of peripheral nerves. Defects in these Schwann cells injury response potentially contribute to the pathogenesis of diabetic peripheral neuropathy (DPN), a common complication of diabetes mellitus. The protein p66shc is essential in regulating oxidative stress responses, autophagy induction and cell survival, and is also vital in the development of DPN. In this study, we hypothesized that p66shc mediates high glucose-induced oxidative stress and autophagic dysfunction. In Schwann cells treated with high glucose; p66shc expression, levels of reactive oxygen species, autophagy impairment, and early apoptosis were elevated. Inhibition of p66shc gene expression by siRNA reversed high glucose-induced oxidative stress, autophagy impairment, and early apoptosis. We also demonstrated that the levels of p66shc was increased, while autophagy-related proteins p62 and LC3 (LC3-II/I) were suppressed in the sciatic nerve of streptozotocin-induced diabetes mice. P66shc-deficient mice exhibited the improvement in autophagy impairment after diabetes onset. Our findings suggest that the p66 plays a crucial role in Schwann cell dysfunction, identifying its potential as a therapeutic target.

Keyword

Autophagy; Diabetic peripheral neuropathy; Oxidative stress; p66shc; Schwann cells

Figure

• 

  Fig. 1 p66shc-deficient Schwann cells attenuated oxidative stress, and early apoptosis triggered by high glucose concentrations. S16 rat Schwann cells were treated with glucose for 72 h. (A) Percentages of apoptotic S16 rat Schwann cells by flow cytometry. (B) p66shc protein expression was measured by Western blotting. (C) Schwann cells transfected with siCON and sip66shc and treated with glucose (25 or 150 mM). ROS was measured by DCF-DA fluorescence and flow cytometry. (D) Percentages of apoptotic S16 rat Schwann cells by flow cytometry. β-actin was used as the loading control. Data represent means ± SEM, n = 3. ROS, reactive oxygen species; PI, propidium iodide; DCF-DA, 2,7- dichlorofluorescein. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, siCON + glucose 150 mM compared with siCON + glucose 25 mM; #p < 0.05, sip66shc + glucose 150 mM compared with siCON + glucose 150 mM.

• 

  Fig. 2 Downregulation of p66shc enhanced the expression of autophagy-related protein in high glucose condition. Schwann cells transfected with siCON and sip66shc and treated with glucose (25 or 150 mM). (A) Expression of autophagy-related proteins (p62 and LC3-II) and p66shc by Western blotting in S16 rat Schwann cells. (B) Immunofluorescence images showing expression of p62 (red) or LC3 (red), and p66shc (green). (C) p62 and LC3 intensity was quantified per cell. Nuclei were stained with DAPI. Scale bar, 20 μm. β-actin was used as the loading control. Data represent means ± SEM, n = 3. DAPI, 4,6-diamidino-2-phenylindole. **p < 0.01 and ***p < 0.001, siCON + glucose 150 mM compared with siCON + glucose 25 mM; #p < 0.05, ##p < 0.01, and ###p < 0.001, sip66shc + glucose 150 mM compared with siCON + glucose 150 mM.

• 

  Fig. 3 Expression of p66shc in the sciatic nerve from STZ-induced diabetes mice. (A, B) Expression of p66shc by Western blotting and immunofluorescence staining, respectively in sciatic nerves from CON and T1DM mice. β-actin was used as the loading control. Nuclei were stained with DAPI. Scale bar, 20 μm. Data represent means ± SEM, n = 15. STZ, streptozotocin; CON, control (saline); T1DM, type 1 diabetes; DAPI, 4,6-diamidino-2-phenylindole. **p < 0.01 and ***p < 0.001.

• 

  Fig. 4 Effect of p66shc KO on autophagy in the sciatic nerve of STZ-induced diabetes mice. (A) Expression of autophagy-related proteins (p62, and LC3-II) by Western blotting in sciatic nerves from CON, T1DM, p66shc KO, and p66shc KO + T1DM mice. β-actin was used as the loading control. (B) Immunofluorescence of the expression of the autophagy-related proteins p62 (red), or LC3 (red), and p66shc (green) in sciatic nerves from CON, T1DM, p66shc KO, and p66shc KO + T1DM mice. (C) The fluorescent area of p62 and LC3 was quantified. Nuclei were stained with DAPI. Scale bar, 20 μm. Data represent means ± SEM, n = 15. CON, control (saline); T1DM, type 1 diabetes mice; p66shc KO, p66shc knockout mice; p66shc KO + T1DM, p66shc KO type 1 diabetic mice; DAPI, 4,6-diamidino-2-phenylindole. *p < 0.05 and ***p < 0.001, T1DM compared with CON; #p < 0.05, ##p < 0.01, and ###p < 0.001, p66shc KO + T1DM compared with T1DM.

Reference

1. Salzer JL. 2015; Schwann cell myelination. Cold Spring Harb Perspect Biol. 7:a020529. DOI: 10.1101/cshperspect.a020529. PMID: 26054742. PMCID: PMC4526746.

2. Wong KM, Babetto E, Beirowski B. 2017; Axon degeneration: make the Schwann cell great again. Neural Regen Res. 12:518–524. DOI: 10.4103/1673-5374.205000. PMID: 28553320. PMCID: PMC5436338.

3. Liu YP, Shao SJ, Guo HD. 2020; Schwann cells apoptosis is induced by high glucose in diabetic peripheral neuropathy. Life Sci. 248:117459. DOI: 10.1016/j.lfs.2020.117459. PMID: 32092332.

4. Feldman EL, Nave KA, Jensen TS, Bennett DLH. 2017; New horizons in diabetic neuropathy: mechanisms, bioenergetics, and pain. Neuron. 93:1296–1313. DOI: 10.1016/j.neuron.2017.02.005. PMID: 28334605. PMCID: PMC5400015.

5. Gonçalves NP, Vægter CB, Andersen H, Østergaard L, Calcutt NA, Jensen TS. 2017; Schwann cell interactions with axons and microvessels in diabetic neuropathy. Nat Rev Neurol. 13:135–147. DOI: 10.1038/nrneurol.2016.201. PMID: 28134254. PMCID: PMC7391875.

6. Hale AN, Ledbetter DJ, Gawriluk TR, Rucker EB 3rd. 2013; Autophagy: regulation and role in development. Autophagy. 9:951–972. DOI: 10.4161/auto.24273. PMID: 24121596. PMCID: PMC3722331.

7. Parzych KR, Klionsky DJ. 2014; An overview of autophagy: morphology, mechanism, and regulation. Antioxid Redox Signal. 20:460–473. DOI: 10.1089/ars.2013.5371. PMID: 23725295. PMCID: PMC3894687.

8. Rodríguez-Vargas JM, Ruiz-Magaña MJ, Ruiz-Ruiz C, Majuelos-Melguizo J, Peralta-Leal A, Rodríguez MI, Muñoz-Gámez JA, de Almodóvar MR, Siles E, Rivas AL, Jäättela M, Oliver FJ. 2012; ROS-induced DNA damage and PARP-1 are required for optimal induction of starvation-induced autophagy. Cell Res. 22:1181–1198. DOI: 10.1038/cr.2012.70. PMID: 22525338. PMCID: PMC3391023.

9. Santana-Codina N, Mancias JD, Kimmelman AC. 2017; The role of autophagy in cancer. Annu Rev Cancer Biol. 1:19–39. DOI: 10.1146/annurev-cancerbio-041816-122338. PMID: 31119201. PMCID: PMC6527373.

10. Kesidou E, Lagoudaki R, Touloumi O, Poulatsidou KN, Simeonidou C. 2013; Autophagy and neurodegenerative disorders. Neural Regen Res. 8:2275–2283.

11. Sciarretta S, Maejima Y, Zablocki D, Sadoshima J. 2018; The role of autophagy in the heart. Annu Rev Physiol. 80:1–26. DOI: 10.1146/annurev-physiol-021317-121427. PMID: 29068766.

12. Ke PY. 2019; Diverse functions of autophagy in liver physiology and liver diseases. Int J Mol Sci. 20:300. DOI: 10.3390/ijms20020300. PMID: 30642133. PMCID: PMC6358975.

13. Djajadikerta A, Keshri S, Pavel M, Prestil R, Ryan L, Rubinsztein DC. 2020; Autophagy induction as a therapeutic strategy for neurodegenerative diseases. J Mol Biol. 432:2799–2821. DOI: 10.1016/j.jmb.2019.12.035. PMID: 31887286.

14. Bhattacharya D, Mukhopadhyay M, Bhattacharyya M, Karmakar P. 2018; Is autophagy associated with diabetes mellitus and its complications? A review. EXCLI J. 17:709–720.

15. Qu L, Liang X, Gu B, Liu W. 2014; Quercetin alleviates high glucose-induced Schwann cell damage by autophagy. Neural Regen Res. 9:1195–1203. DOI: 10.4103/1673-5374.135328. PMID: 25206782. PMCID: PMC4146282.

16. Chung YC, Lim JH, Oh HM, Kim HW, Kim MY, Kim EN, Kim Y, Chang YS, Kim HW, Park CW. 2018; Calcimimetic restores diabetic peripheral neuropathy by ameliorating apoptosis and improving autophagy. Cell Death Dis. 9:1163. DOI: 10.1038/s41419-018-1192-7. PMID: 30478254. PMCID: PMC6255917.

17. Migliaccio E, Mele S, Salcini AE, Pelicci G, Lai KM, Superti-Furga G, Pawson T, Di Fiore PP, Lanfrancone L, Pelicci PG. 1997; Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway. EMBO J. 16:706–716. DOI: 10.1093/emboj/16.4.706. PMID: 9049300. PMCID: PMC1169672.

18. Pagnin E, Fadini G, de Toni R, Tiengo A, Calò L, Avogaro A. 2005; Diabetes induces p66shc gene expression in human peripheral blood mononuclear cells: relationship to oxidative stress. J Clin Endocrinol Metab. 90:1130–1136. DOI: 10.1210/jc.2004-1283. PMID: 15562031.

19. Menini S, Amadio L, Oddi G, Ricci C, Pesce C, Pugliese F, Giorgio M, Migliaccio E, Pelicci P, Iacobini C, Pugliese G. 2006; Deletion of p66Shc longevity gene protects against experimental diabetic glomerulopathy by preventing diabetes-induced oxidative stress. Diabetes. 55:1642–1650. Erratum. DOI: 10.2337/db18-er01a. PMID: 29066598. PMCID: PMC5741146.

20. Li Q, Kim YR, Vikram A, Kumar S, Kassan M, Gabani M, Lee SK, Jacobs JS, Irani K. 2016; P66Shc-induced microRNA-34a causes diabetic endothelial dysfunction by downregulating sirtuin1. Arterioscler Thromb Vasc Biol. 36:2394–2403. DOI: 10.1161/ATVBAHA.116.308321. PMID: 27789474. PMCID: PMC5293179.

21. Zhou S, Chen HZ, Wan YZ, Zhang QJ, Wei YS, Huang S, Liu JJ, Lu YB, Zhang ZQ, Yang RF, Zhang R, Cai H, Liu DP, Liang CC. 2011; Repression of P66Shc expression by SIRT1 contributes to the prevention of hyperglycemia-induced endothelial dysfunction. Circ Res. 109:639–648. DOI: 10.1161/CIRCRESAHA.111.243592. PMID: 21778425.

22. Camici GG, Schiavoni M, Francia P, Bachschmid M, Martin-Padura I, Hersberger M, Tanner FC, Pelicci P, Volpe M, Anversa P, Lüscher TF, Cosentino F. 2007; Genetic deletion of p66(Shc) adaptor protein prevents hyperglycemia-induced endothelial dysfunction and oxidative stress. Proc Natl Acad Sci U S A. 104:5217–5222. DOI: 10.1073/pnas.0609656104. PMID: 17360381. PMCID: PMC1829289.

23. Mishra M, Duraisamy AJ, Bhattacharjee S, Kowluru RA. 2019; Adaptor protein p66Shc: a link between cytosolic and mitochondrial dysfunction in the development of diabetic retinopathy. Antioxid Redox Signal. 30:1621–1634. DOI: 10.1089/ars.2018.7542. PMID: 30105917. PMCID: PMC6459280.

24. Al Sabaani N. 2021; Exendin-4 inhibits high glucose-induced oxidative stress in retinal pigment epithelial cells by modulating the expression and activation of p₆₆Shc. Cutan Ocul Toxicol. 40:175–186. DOI: 10.1080/15569527.2020.1844727. PMID: 34275397.

25. Onnis A, Cianfanelli V, Cassioli C, Samardzic D, Pelicci PG, Cecconi F, Baldari CT. 2018; The pro-oxidant adaptor p66SHC promotes B cell mitophagy by disrupting mitochondrial integrity and recruiting LC3-II. Autophagy. 14:2117–2138. DOI: 10.1080/15548627.2018.1505153. PMID: 30109811. PMCID: PMC6984773.

26. Krijnen PA, Simsek S, Niessen HW. 2009; Apoptosis in diabetes. Apoptosis. 14:1387–1388. DOI: 10.1007/s10495-009-0419-6. PMID: 19856207. PMCID: PMC2773035.

27. Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC. 2003; Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes. 52:102–110. DOI: 10.2337/diabetes.52.1.102. PMID: 12502499.

28. Volpe CMO, Villar-Delfino PH, Dos Anjos PMF, Nogueira-Machado JA. 2018; Cellular death, reactive oxygen species (ROS) and diabetic complications. Cell Death Dis. 9:119. DOI: 10.1038/s41419-017-0135-z. PMID: 29371661. PMCID: PMC5833737.

29. Galimov ER. 2010; The role of p66shc in oxidative stress and apoptosis. Acta Naturae. 2:44–51. DOI: 10.32607/20758251-2010-2-4-44-51. PMID: 22649663. PMCID: PMC3347587.

30. Turkmen K. 2017; Inflammation, oxidative stress, apoptosis, and autophagy in diabetes mellitus and diabetic kidney disease: the Four Horsemen of the Apocalypse. Int Urol Nephrol. 49:837–844. DOI: 10.1007/s11255-016-1488-4. PMID: 28035619.

31. Choi SJ, Kim S, Lee WS, Kim DW, Kim CS, Oh SH. 2023; Autophagy dysfunction in a diabetic peripheral neuropathy model. Plast Reconstr Surg. 151:355–364. DOI: 10.1097/PRS.0000000000009844.

32. Liu WJ, Ye L, Huang WF, Guo LJ, Xu ZG, Wu HL, Yang C, Liu HF. 2016; p62 links the autophagy pathway and the ubiqutin-proteasome system upon ubiquitinated protein degradation. Cell Mol Biol Lett. 21:29. DOI: 10.1186/s11658-016-0031-z. PMID: 28536631. PMCID: PMC5415757.

33. Edwards JL, Vincent AM, Cheng HT, Feldman EL. 2008; Diabetic neuropathy: mechanisms to management. Pharmacol Ther. 120:1–34. DOI: 10.1016/j.pharmthera.2008.05.005. PMID: 18616962. PMCID: PMC4007052.

34. Gonçalves NP, Vægter CB, Pallesen LT. 2018; Peripheral glial cells in the development of diabetic neuropathy. Front Neurol. 9:268. DOI: 10.3389/fneur.2018.00268. PMID: 29770116. PMCID: PMC5940740.

35. Fadini GP, Albiero M, Bonora BM, Poncina N, Vigili de Kreutzenberg S, Avogaro A. 2018; p66Shc gene expression in peripheral blood mononuclear cells and progression of diabetic complications. Cardiovasc Diabetol. 17:16. DOI: 10.1186/s12933-018-0660-9. PMID: 29343271. PMCID: PMC5771224.

36. Towns R, Kabeya Y, Yoshimori T, Guo C, Shangguan Y, Hong S, Kaplan M, Klionsky DJ, Wiley JW. 2005; Sera from patients with type 2 diabetes and neuropathy induce autophagy and colocalization with mitochondria in SY5Y cells. Autophagy. 1:163–170. DOI: 10.4161/auto.1.3.2068. PMID: 16874076.

37. Yang S, Xia C, Li S, Du L, Zhang L, Hu Y. 2014; Mitochondrial dysfunction driven by the LRRK2-mediated pathway is associated with loss of Purkinje cells and motor coordination deficits in diabetic rat model. Cell Death Dis. 5:e1217. DOI: 10.1038/cddis.2014.184. PMID: 24810053. PMCID: PMC4047887.

38. Tanida I, Ueno T, Kominami E. 2004; LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol. 36:2503–2518. DOI: 10.1016/j.biocel.2004.05.009. PMID: 15325588. PMCID: PMC7129593.

39. Filomeni G, De Zio D, Cecconi F. 2015; Oxidative stress and autophagy: the clash between damage and metabolic needs. Cell Death Differ. 22:377–388. DOI: 10.1038/cdd.2014.150. PMID: 25257172. PMCID: PMC4326572.

40. Talebi M, Mohammadi Vadoud SA, Haratian A, Talebi M, Farkhondeh T, Pourbagher-Shahri AM, Samarghandian S. 2022; The interplay between oxidative stress and autophagy: focus on the development of neurological diseases. Behav Brain Funct. 18:3. DOI: 10.1186/s12993-022-00187-3. PMID: 35093121. PMCID: PMC8799983.

41. Ning S, Wang L. 2019; The multifunctional protein p62 and its mechanistic roles in cancers. Curr Cancer Drug Targets. 19:468–478. DOI: 10.2174/1568009618666181016164920. PMID: 30332964. PMCID: PMC8052633.

42. Lin JH, Walter P, Yen TS. 2008; Endoplasmic reticulum stress in disease pathogenesis. Annu Rev Pathol. 3:399–425. DOI: 10.1146/annurev.pathmechdis.3.121806.151434. PMID: 18039139. PMCID: PMC3653419.

43. Inceoglu B, Bettaieb A, Trindade da Silva CA, Lee KS, Haj FG, Hammock BD. 2015; Endoplasmic reticulum stress in the peripheral nervous system is a significant driver of neuropathic pain. Proc Natl Acad Sci U S A. 112:9082–9087. DOI: 10.1073/pnas.1510137112. PMID: 26150506. PMCID: PMC4517273.

44. Ogata M, Hino S, Saito A, Morikawa K, Kondo S, Kanemoto S, Murakami T, Taniguchi M, Tanii I, Yoshinaga K, Shiosaka S, Hammarback JA, Urano F, Imaizumi K. 2006; Autophagy is activated for cell survival after endoplasmic reticulum stress. Mol Cell Biol. 26:9220–9231. DOI: 10.1128/MCB.01453-06. PMID: 17030611. PMCID: PMC1698520.

45. Rashid HO, Yadav RK, Kim HR, Chae HJ. 2015; ER stress: autophagy induction, inhibition and selection. Autophagy. 11:1956–1977. DOI: 10.1080/15548627.2015.1091141. PMID: 26389781. PMCID: PMC4824587.

46. Su H, Wang X. 2011; p62 stages an interplay between the ubiquitin-proteasome system and autophagy in the heart of defense against proteotoxic stress. Trends Cardiovasc Med. 21:224–228. DOI: 10.1016/j.tcm.2012.05.015. PMID: 22902070. PMCID: PMC3424486.

47. Bhandary B, Marahatta A, Kim HR, Chae HJ. 2012; An involvement of oxidative stress in endoplasmic reticulum stress and its associated diseases. Int J Mol Sci. 14:434–456. DOI: 10.3390/ijms14010434. PMID: 23263672. PMCID: PMC3565273.

48. Rahman MA, Hannan MA, Dash R, Rahman MH, Islam R, Uddin MJ, Sohag AAM, Rahman MH, Rhim H. 2021; Phytochemicals as a complement to cancer chemotherapy: pharmacological modulation of the autophagy-apoptosis pathway. Front Pharmacol. 12:639628. DOI: 10.3389/fphar.2021.639628. PMID: 34025409. PMCID: PMC8138161.

49. Ortega-Camarillo C, Guzmán-Grenfell AM, García-Macedo R, Rosales-Torres AM, Avalos-Rodríguez A, Durán-Reyes G, Medina-Navarro R, Cruz M, Díaz-Flores M, Kumate J. 2006; Hyperglycemia induces apoptosis and p53 mobilization to mitochondria in RINm5F cells. Mol Cell Biochem. 281:163–171. DOI: 10.1007/s11010-006-0829-5. PMID: 16328969.

50. Kumar S, Kain V, Sitasawad SL. 2012; High glucose-induced Ca2+ overload and oxidative stress contribute to apoptosis of cardiac cells through mitochondrial dependent and independent pathways. Biochim Biophys Acta. 1820:907–920. DOI: 10.1016/j.bbagen.2012.02.010. PMID: 22402252.

51. Zhou F, Yang Y, Xing D. 2011; Bcl-2 and Bcl-xL play important roles in the crosstalk between autophagy and apoptosis. FEBS J. 278:403–413. DOI: 10.1111/j.1742-4658.2010.07965.x. PMID: 21182587.

52. Su M, Mei Y, Sinha S. 2013; Role of the crosstalk between autophagy and apoptosis in cancer. J Oncol. 2013:102735. DOI: 10.1155/2013/102735. PMID: 23840208. PMCID: PMC3687500.