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Current Pharmaceutical Design

Author(s): Jiangxia Chen, Lijun Zhou, Qingchun Zhao* and Zhentong Qi

DOI: 10.2174/0113816128278324240115104615

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

Page: [552 - 563] Pages: 12

  • * (Excluding Mailing and Handling)

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.

[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.
[http://dx.doi.org/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]
2023 Alzheimer’s disease facts and figures. Alzheimers Dement 2023; 19(4): 1598-695.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/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.
[http://dx.doi.org/10.1016/j.ejmech.2023.115145] [PMID: 36706620]