A New Cell Model Overexpressing sTGFBR3 for Studying Alzheimer's Disease In vitro


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Abstract

Background::Recent studies have suggested that abnormal microglial hyperactivation has an important role in the progression of Alzheimer's disease (AD). sTGFBR3 (a shed extracellular domain of the transforming growth factor type III receptor) is a newly identified target of microglia polarization dysregulation, whose overexpression can cause abnormal accumulation of transforming growth factor β1 (TGF-β1), promoting Aβ, tau, and neuroinflammatory pathology.

Objective::The objective of this study is to develop and validate a new cell model overexpressing sTGFBR3 for studying AD in vitro.

Methods::BV2 cells (a microglial cell derived from C57/BL6 murine) were used as a cell model. Cells were then treated with different concentrations of lipopolysaccharide (LPS) (0, 1, or 0.3 µg/mL) for 12, 24, or 48h and then with or without sodium pervanadate (100 µM) for 30 min. Next, the effect surface optimization method was used to determine optimal experimental conditions. Finally, the optimized model was used to assess the effect of ZQX series compounds and vasicine on cell viability and protein expression. Expression of TGFBR3 and TNF-α was assessed using Western blot. MTT assay was used to assess cell viability, and enzyme- linked immunosorbent assay (ELISA) was employed to evaluate extracellular TGF-β1 and sTGFBR3

Results::LPS (0.3 µg/mL) treatment for 11 h at a cell density of 60% and pervanadate concentration (100 µM) incubation for 30 min were the optimal experimental conditions for increasing membrane protein TGFBR3 overexpression, as well as extracellular sTGFBR3 and TGF-β1. Applying ZQX-5 and vasicine reversed this process by reducing extracellular TGF-β1, promoting the phosphorylation of Smad2/3, a protein downstream of TGF-β1, and inhibiting the release of the inflammatory factor TNF-α.

Conclusion::This new in vitro model may be a useful cell model for studying Alzheimer's disease in vitro

About the authors

Jiangxia Chen

General Hospital of Northern Theatre Command, Bei Fang Hospital of Shenyang Pharmaceutical University

Email: info@benthamscience.net

Lijun Zhou

General Hospital of Northern Theatre Command, Bei Fang Hospital of Shenyang Pharmaceutical University

Email: info@benthamscience.net

Qingchun Zhao

General Hospital of Northern Theatre Command,, Bei Fang Hospital of Shenyang Pharmaceutical Universit

Author for correspondence.
Email: info@benthamscience.net

Zhentong Qi

General Hospital of Northern Theatre Command, Bei Fang Hospital of Shenyang Pharmaceutical University

Email: info@benthamscience.net

References

  1. Nozhat Z, Khalaji MS, Hedayati M, Kia SK. Different methods for cell viability and proliferation assay: Essential tools in pharmaceutical studies. Anticancer Agents Med Chem 2022; 22(4): 703-12. doi: 10.2174/1871520621999201230202614 PMID: 33390140
  2. Weller J, Budson A. Current understanding of Alzheimer’s disease diagnosis and treatment. F1000 Res 2018; 7: 2046-1402.
  3. Alzheimer’s disease facts and figures. Alzheimers Dement 2023; 19(4): 1598-695. doi: 10.1002/alz.13016 PMID: 36918389
  4. Yiannopoulou KG, Papageorgiou SG. Current and future treatments in Alzheimer disease: An update. J Cent Nerv Syst Dis 2020; 12: 1179573520907397. doi: 10.1177/1179573520907397 PMID: 32165850
  5. Boskabadi H, Zakerihamidi M, Moradi A. Predictive value of biochemical and hematological markers in prognosis of asphyxic infants. Caspian J Intern Med 2020; 11(4): 377-83. PMID: 33680378
  6. Sierra A, Paolicelli RC, Kettenmann H. Cien años de microglía: Milestones in a century of microglial research. Trends Neurosci 2019; 42(11): 778-92. doi: 10.1016/j.tins.2019.09.004 PMID: 31635851
  7. Tay TL, Béchade C, D’Andrea I, et al. Microglia gone rogue: Impacts on psychiatric disorders across the lifespan. Front Mol Neurosci 2018; 10: 421. doi: 10.3389/fnmol.2017.00421 PMID: 29354029
  8. d’Errico P, Ziegler-Waldkirch S, Aires V, et al. Microglia contribute to the propagation of Aβ into unaffected brain tissue. Nat Neurosci 2022; 25(1): 20-5. doi: 10.1038/s41593-021-00951-0 PMID: 34811521
  9. Hansen DV, Hanson JE, Sheng M. Microglia in Alzheimer’s disease. J Cell Biol 2018; 217(2): 459-72. doi: 10.1083/jcb.201709069 PMID: 29196460
  10. Uddin MS, Lim LW. Glial cells in Alzheimer’s disease: From neuropathological changes to therapeutic implications. Ageing Res Rev 2022; 78: 101-622.
  11. Chun H, Marriott I, Lee CJ, Cho H. Elucidating the interactive roles of glia in Alzheimer’s disease using established and newly developed experimental models. Front Neurol 2018; 9: 1664-2295.
  12. Qin Q, Teng Z, Liu C, Li Q, Yin Y, Tang Y. TREM2, microglia, and Alzheimer’s disease. Mech Ageing Dev 2021; 195: 111-438.
  13. Guo S, Wang H, Yin Y. Microglia polarization from M1 to M2 in neurodegenerative diseases. Front Aging Neurosci 2022; 14: 815347. doi: 10.3389/fnagi.2022.815347 PMID: 35250543
  14. Tang Y, Le W. Differential roles of M1 and M2 microglia in neurodegenerative diseases. Mol Neurobiol 2016; 53(2): 1181-94. doi: 10.1007/s12035-014-9070-5 PMID: 25598354
  15. Yao K, Zu H. Microglial polarization: Novel therapeutic mechanism against Alzheimer’s disease. Inflammopharmacology 2020; 28(1): 95-110. doi: 10.1007/s10787-019-00613-5 PMID: 31264132
  16. Wang Q, Yao H, Liu W, et al. Microglia polarization in Alzheimer’s disease: Mechanisms and a potential therapeutic target. Front Aging Neurosci 2021; 13: 7727-17.
  17. Nimmerjahn A, Kirchhoff F, Helmchen F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 2005; 308(5726): 1314-8. doi: 10.1126/science.1110647 PMID: 15831717
  18. Yuan Y, Wu C, Ling EA. Heterogeneity of microglia phenotypes: Developmental, functional and some therapeutic considerations. Curr Pharm Des 2019; 21: 2375-93.
  19. Du L, Zhang Y, Chen Y, Zhu J, Yang Y, Zhang HA-O. Role of microglia in neurological disorders and their potentials as a therapeutic target. Mol Neurobiol 2017; 10: 7567-84.
  20. Zhou C, Li JX, Zheng CX, et al. Neuroprotective effects of Jie-duhuo- xue decoction on microglia pyroptosis after cerebral ischemia and reperfusion-from the perspective of glial-vascular unit. J Ethnopharmacol 2024; 318(Pt B): 116-990.
  21. Subramaniam SR, Federoff HJ. Targeting microglial activation states as a therapeutic avenue in Parkinson’s disease. Front Aging Neurosci 2017; 9: 176.
  22. Wang Q, Yao H, Liu W, et al. Microglia polarization in Alzheimer’s disease: Mechanisms and a potential therapeutic target. Front Aging Neurosci 2021; 13: 772717. doi: 10.3389/fnagi.2021.772717 PMID: 34819850
  23. Khan S, Barve KH, Kumar MS. Recent advancements in pathogenesis, diagnostics and treatment of Alzheimer’s disease. Curr Neuropharmacol 2020; 18(11): 1106-25. doi: 10.2174/1570159X18666200528142429 PMID: 32484110
  24. Ashrafian H, Zadeh EH, Khan RH. Review on Alzheimer’s disease: Inhibition of amyloid beta and tau tangle formation. Int J Biol Macromol 2021; 167: 382-94.
  25. Tiwari S, Atluri V, Kaushik A, Yndart A, Nair M. Alzheimer’s disease: Pathogenesis, diagnostics, and therapeutics. Int J Nanomed 2019; 14: 5541-54.
  26. Yunna C, Mengru H, Lei W, Weidong C. Macrophage M1/M2 polarization. Eur J Pharmacol 2020; 877: 1730-90.
  27. Zheng M, Zhu Y, Wei KAO, et al. Metformin attenuates the inflammatory response via the regulation of synovial m1 macrophage in osteoarthritis. Int J Mol Sci 2023; 6: 5355.
  28. Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH. Mechanisms underlying inflammation in neurodegeneration. Cell 2010; 140(6): 918-34. doi: 10.1016/j.cell.2010.02.016 PMID: 20303880
  29. Ising C, Venegas C, Zhang S, et al. NLRP3 inflammasome activation drives tau pathology. Nature 2019; 575(7784): 669-73. doi: 10.1038/s41586-019-1769-z PMID: 31748742
  30. Leng F, Edison P. Neuroinflammation and microglial activation in Alzheimer disease: Where do we go from here? Nat Rev Neurol 2021; 17(3): 157-72. doi: 10.1038/s41582-020-00435-y PMID: 33318676
  31. Villarreal MM, Kim SK, Barron L, et al. Correction to binding properties of the transforming growth factor-β coreceptor betaglycan: Proposed mechanism for potentiation of receptor complex assembly and signaling. Biochemistry 2017; 56(28): 3689. doi: 10.1021/acs.biochem.7b00566 PMID: 28677957
  32. Knelson EH, Nee JC, Blobe GC. Heparan sulfate signaling in cancer. Trends Biochem Sci 2014; 39(6): 277-88. doi: 10.1016/j.tibs.2014.03.001 PMID: 24755488
  33. Quan X, Liang H, Chen Y, Qin Q, Wei Y, Liang Z. Related network and differential expression analyses identify nuclear genes and pathways in the hippocampus of Alzheimer disease. Med Sci Monit 2020; 26: e919311.
  34. Py NA, Bonnet AE, Bernard A, et al. Differential spatio-temporal regulation of MMPs in the 5xFAD mouse model of Alzheimer’s disease: Evidence for a pro-amyloidogenic role of MT1-MMP. Front Aging Neurosci 2014; 6: 247. doi: 10.3389/fnagi.2014.00247 PMID: 25278878
  35. Velasco-Loyden G, Arribas J, López-Casillas F. The shedding of betaglycan is regulated by pervanadate and mediated by membrane type matrix metalloprotease-1. J Biol Chem 2004; 279(9): 7721-33. doi: 10.1074/jbc.M306499200 PMID: 14672946
  36. Liu Y, Aguzzi A. NG2 glia are required for maintaining microglia homeostatic state. Glia 2020; 68(2): 345-55. doi: 10.1002/glia.23721 PMID: 31518022
  37. Zhang S, Wang Q, Yang Q, et al. NG2 glia regulate brain innate immunity via TGF-β2/TGFBR2 axis. BMC Med 2019; 17(1): 204. doi: 10.1186/s12916-019-1439-x PMID: 31727112
  38. Pál G, Vincze C, Renner É, et al. Time course, distribution and cell types of induction of transforming growth factor betas following middle cerebral artery occlusion in the rat brain. PLoS One 2012; 7(10): e46731. doi: 10.1371/journal.pone.0046731 PMID: 23056426
  39. Forsey RJ, Thompson JM, Ernerudh J, et al. Plasma cytokine profiles in elderly humans. Mech Ageing Dev 2003; 124(4): 487-93. doi: 10.1016/S0047-6374(03)00025-3 PMID: 12714257
  40. Carrieri G, Marzi E, Olivieri F, et al. The G/C915 polymorphism of transforming growth factor β1 is associated with human longevity: A study in Italian centenarians. Aging Cell 2004; 3(6): 443-8. doi: 10.1111/j.1474-9728.2004.00129.x PMID: 15569360
  41. Heldin CH, Moustakas A. Signaling receptors for tgf-β family members. Cold Spring Harb Perspect Biol 2016; 8(8): a022053. doi: 10.1101/cshperspect.a022053 PMID: 27481709
  42. Vander Ark A, Cao J, Li X. TGF-β receptors: In and beyond TGF-β signaling. Cell Signal 2018; 52: 112-20. doi: 10.1016/j.cellsig.2018.09.002 PMID: 30184463
  43. Meng X, Kuang H, Wang Q, Zhang H, Wang D, Kang T. A polysaccharide from Codonopsis pilosula roots attenuates carbon tetrachloride-induced liver fibrosis via modulation of TLR4/NF-κB and TGF-β1/Smad3 signaling pathway. Int Immunopharmacol 2023; 119: 1878-705.
  44. Ying H, Fang M, Hang QQ, Chen Y, Qian X, Chen M. Pirfenidone modulates macrophage polarization and ameliorates radiation-induced lung fibrosis by inhibiting the TGF-β1/Smad3 pathway. J Cell Mol Med 2021; 25(18): 8662-75. doi: 10.1111/jcmm.16821 PMID: 34327818
  45. Yeh YY, Chiao CC, Kuo WY, et al. TGF-β1 increases motility and αvβ3 integrin up-regulation via PI3K, Akt and NF-κB-dependent pathway in human chondrosarcoma cells. Biochem Pharmacol 2008; 75(6): 1292-301. doi: 10.1016/j.bcp.2007.11.017 PMID: 18191107
  46. Ślusarczyk J, Trojan E, Głombik K, et al. Targeting the NLRP3 inflammasome-related pathways via tianeptine treatment-suppressed microglia polarization to the M1 phenotype in lipopolysaccharide-stimulated cultures. Int J Mol Sci 2018; 19(7): 1965. doi: 10.3390/ijms19071965 PMID: 29976873
  47. Wang H, Liu C, Han M, Cheng C, Zhang D. TRAM1 promotes microglia M1 polarization. J Mol Neurosci 2016; 58(2): 287-96. doi: 10.1007/s12031-015-0678-3 PMID: 26563450
  48. Niyatee S, Lane-Donovan C, VandeVrede L, Adam LB. Tau pathology in neurodegenerative disease: Disease mechanisms and therapeutic avenues. J Clin Invest 2023; 133(12): e168553.
  49. Zhang F, Zhong R, Li S, et al. Acute hypoxia induced an imbalanced M1/M2 activation of microglia through NF-κB signaling in Alzheimer’s disease mice and wild-type littermates. Front Aging Neurosci 2017; 9: 282.
  50. Chu W. TGFBR3, a potential negative regulator of TGF-β signaling, protects cardiac fibroblasts from hypoxia-induced apoptosis. J Cell Physiol 2011; 10: 1097-4652.
  51. Philippeos C, Hughes RD, Dhawan A, Mitry RR. Introduction to cell culture. Methods Mol Biol 2012; 806: 1940-6029.
  52. Baust JM, Buehring GC, Campbell L, et al. Best practices in cell culture: An overview. In Vitro Cell Dev Biol Anim 2017; 53(8): 669-72. doi: 10.1007/s11626-017-0177-7 PMID: 28808859
  53. Zhao Y, Jaber VR, Pogue AI, Sharfman NM, Taylor C, Lukiw WJ. Lipopolysaccharides (LPSs) as potent neurotoxic glycolipids in Alzheimer’s disease (AD). Int J Mol Sci 2022; 23(20): 12671.
  54. Narenderan ST, Meyyanathan SN, Karri V. Experimental design in pesticide extraction methods: A review. Food Chem 2019; 289: 384-95.
  55. Bezerra MA, Ricardo ES, Eliane PO, Leonardo SV, Luciane AE. Escaleira, response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta 2008; 76: 965-77.
  56. Tabatabaei MS, Ahmed M. Enzyme-linked immunosorbent assay (ELISA). Methods Mol Biol 2022; 2508: 115-34.
  57. Aydin S. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides 2015; 72: 4-15.
  58. Reen DJ. Enzyme-linked immunosorbent assay (ELISA). Methods Mol Biol 1994; 32: 461-6.
  59. Hnasko TS, Hnasko RM. The western blot. Methods Mol Biol 2015; 1318: 87-96.
  60. Priti K, Arvindhan N, Pradeep DU. Analysis of cell viability by the MTT assay. Cold Spring Harb Protoc 2018; 2018: 6.
  61. Präbst K, Engelhardt H, Ringgeler S, Hübner H. Basic colorimetric proliferation assays: MTT, WST, and resazurin. Methods Mol Biol 2017; 1601: 1-17.
  62. Pillai-Kastoori L, Schutz-Geschwender AR, Harford JA. A systematic approach to quantitative Western blot analysis. Anal Biochem 2020; 593: 113-608.
  63. Hirano S. Western blot analysis. Methods Mol Biol 2012; 926: 87-97.
  64. Kim B. Western blot techniques. Methods Mol Biol 2017; 1606: 133-9.
  65. Taylor SC, Posch A. The design of a quantitative western blot experiment. Biomed Res Int 2014; 2014: 361-590.
  66. Armstrong RA, Eperjesi F, Gilmartin B. The application of analysis of variance (ANOVA) to different experimental designs in optometry. Ophthalmic Physiol Opt 2002; 22(3): 248-56. doi: 10.1046/j.1475-1313.2002.00020.x PMID: 12090640
  67. Mishra P, Singh U, Pandey C, Mishra P, Pandey G. Application of student’s t-test, analysis of variance, and covariance. Ann Card Anaesth 2019; 22(4): 407-11. doi: 10.4103/aca.ACA_94_19 PMID: 31621677
  68. Chatzi A, Doody O. The one-way ANOVA test explained. Nurse Res 2023; 31(3): 8-14. doi: 10.7748/nr.2023.e1885 PMID: 37317616
  69. Cheng J, Zhang R, Xu Z, et al. Early glycolytic reprogramming controls microglial inflammatory activation. J Neuroinflamm 2021; 18(1): 129. doi: 10.1186/s12974-021-02187-y PMID: 34107997
  70. Hata A, Chen YG. TGF-β signaling from receptors to smads. Cold Spring Harb Perspect Biol 2016; 8(9): a022061. doi: 10.1101/cshperspect.a022061 PMID: 27449815
  71. Spittau B. Transforming growth factor β1-mediated anti-inflammation slows progression of midbrain dopaminergic neurodegeneration in Parkinson′s disease? Neural Regen Res 2015; 10(10): 1578-80. doi: 10.4103/1673-5374.165228 PMID: 26692847
  72. Cai Y, Liu J, Wang B, Sun M, Yang H. Microglia in the neuroinflammatory pathogenesis of Alzheimer’s disease and related therapeutic targets. Front Immunol 2022; 13: 856376.
  73. Ji Z, Liu C, Zhao W, Soto C, Zhou X. Multi-scale modeling for systematically understanding the key roles of microglia in AD development. Comput Biol Med 2021; 133: 104-374.
  74. Wang C, Zong S, Cui X, et al. The effects of microglia-associated neuroinflammation on Alzheimer’s disease. Front Immunol 2023; 14: 1117-72.
  75. Han D, Zhou Z, Liu J, Wang T, Yin J. Neuroprotective effects of isoflurane against lipopolysaccharide-induced neuroinflammation in BV2 microglial cells by regulating HMGB1/TLRs pathway. Folia Neuropathol 2020; 58(1): 57-69. doi: 10.5114/fn.2020.94007 PMID: 32337958
  76. Bhanukiran K. T A G, Krishnamurthy S, Singh SK, Hemalatha S. Discovery of multi-target directed 3-OH pyrrolidine derivatives through a semisynthetic approach from alkaloid vasicine for the treatment of Alzheimer’s disease. Eur J Med Chem 2023; 249: 115145. doi: 10.1016/j.ejmech.2023.115145 PMID: 36706620

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