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The Role of Thionine on Amyloid Precursor Protein Processing in N2a-APPSwe Cells

Year 2025, Volume: 12 Issue: 2, 161 - 167, 28.08.2025
https://doi.org/10.47572/muskutd.1698250

Abstract

Alzheimer's disease, marked by aberrant accumulation of amyloid-β (Aβ) peptides produced by the cleavage of amyloid precursor protein (APP) by secretases, is associated with cognitive impairment. Evidence has shown that cholinergic activity may alter the management of APP processing. The objective of this investigation was to assess the effect of thionine (TH), a phenothiazine-structured cholinesterase inhibitor, on the processing of APP in N2a mouse neuroblastoma cells that overexpress the Swedish mutant of APP (N2a/APPSwe cells). To this end, we investigated how TH affects levels of Aβ40/42 peptides, soluble APPα (sAPPα), intracellular APP, β-site APP-cleaving enzyme 1 (BACE-1), presenilin-1 (PS1), and glycogen synthase kinase 3 beta (GSK3β). After treating N2a/APPSwe cells with TH at concentrations of 1.25-10 μM for 24 h, the levels of Aβ42, Aβ40 and sAPPα were evaluated using ELISA. In addition, the effects of thionine on intracellular APP, PS1 and GSK3β levels were analyzed by Western blotting. Our findings showed that thionine significantly reduced the levels of Aβ peptides and APP in N2a/APPSwe cells. When we evaluated the effects of thionine on APP processing, no significant alteration was detected in the level of BACE-1. However, a statistically significant reduction was observed in the levels of PS1 and GSK3β. The results provide an important mechanism of action for thionine's potential to prevent amyloid pathology.

Supporting Institution

Scientific Research Projects Coordination Unit of Hacettepe University

Project Number

TSA-2019-17288

References

  • Toodayan N. Professor Alois Alzheimer (1864–1915): lest we forget. J Clin Neurosci. 2016;31:47-55.
  • Janoutová J, Kovalová M, Machaczka O, et al. Risk factors for Alzheimer’s disease: an epidemiological study. Curr Alzheimer Res. 2021;18(5):372-9.
  • Dos Santos Picanco LC, Ozela PF, de Fatima de Brito Brito M, et al. Alzheimer's disease: a review from the pathophysiology to diagnosis, new perspectives for pharmacological treatment. Curr Med Chem. 2018;25(26):3141-59.
  • Kang J, Lemaire H-G, Unterbeck A, et al. The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature. 1987;325(6106):733-6.
  • Haass C, Selkoe DJ. Cellular processing of beta-amyloid precursor protein and the genesis of amyloid beta-peptide. Cell. 1993;75(6):1039-42.
  • Chen J, Chen J-S, Li S, Zhang F, et al. Amyloid precursor protein: a regulatory hub in Alzheimer's disease. Aging Dis. 2024;15(1):201.
  • Steiner H, Fukumori A, Tagami S, et al. Making the final cut: pathogenic amyloid-β peptide generation by γ-secretase. Cell Stress. 2018;2(11):292.
  • Ittner LM, Götz J. Amyloid-β and tau—a toxic pas de deux in Alzheimer's disease. Nat Rev Neurosci. 2011;12(2):67-72.
  • Green FJ. The Sigma-Aldrich handbook of stains, dyes, and indicators. 1990.
  • Wainwright M, Phoenix D, Laycock S, et al. Photobactericidal activity of phenothiazinium dyes against methicillin-resistant strains of Staphylococcus aureus. FEMS Microbiol Lett. 1998;160(2):177-81.
  • Harris F, Chatfield L, Phoenix D. Phenothiazinium based photosensitisers-photodynamic agents with a multiplicity of cellular targets and clinical applications. Curr Drug Targets. 2005;6(5):615-27.
  • Gualdesi MS, Vara J, Aiassa V, Igarzabal CA, Ortiz CS. Thionine in the design of new photosensitizers: Bromination and vehiculization in polymeric nanoparticles. J Mol Liq. 2020;310:113247.
  • Yuksel M, Biberoglu K, Onder S, et al. Effects of phenothiazine-structured compounds on APP processing in Alzheimer's disease cellular model. Biochimie. 2017;138:82-9.
  • Onder S, Biberoglu K, Yuksel M, et al. Toluidine blue O attenuates tau phosphorylation in N2a-APPSwe cells. Chem Biol Interact. 2022;366:110126.
  • Biberoglu K, Tek MY, Ghasemi ST, et al. Toluidine blue O is a potent inhibitor of human cholinesterases. Arch Biochem Biophys. 2016;604:57-62.
  • Onder S, Biberoglu K, Yuksel M, et al. Fenotı̇yazı̇n Yapısındakı̇ Kolı̇nesteraz İnhı̇bı̇törlerı̇nı̇n Amı̇loı̇d Prekürsör Proteı̇n Metabolı̇zması Ve Tau Üzerı̇ndekı̇ Etkı̇ Mekanı̇zmalarının Araştırılması, Hacettepe Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi. 2019.
  • Loetscher H, Deuschle U, Brockhaus M, et al. Presenilins are processed by caspase-type proteases. J Biol Chem. 1997;272(33):20655-9.
  • Lauretti E, Dincer O, Praticò D. Glycogen synthase kinase-3 signaling in Alzheimer's disease. Biochim Biophys Acta. 2020;1867(5):118664.
  • Kurkinen M, Fułek M, Fułek K, et al. The amyloid cascade hypothesis in Alzheimer’s disease: should we change our thinking? Biomolecules. 2023;13(3):453.
  • Majdi A, Sadigh-Eteghad S, Rahigh Aghsan, et al. Amyloid-β, tau, and the cholinergic system in Alzheimer’s disease: Seeking direction in a tangle of clues. Rev Neurosci. 2020;31(4):391-413.
  • Haass C, Selkoe DJ. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid β-peptide. Nat Rev Mol Cell Biol. 2007;8(2):101-12.
  • Ricciarelli R, Fedele E. The amyloid cascade hypothesis in Alzheimer's disease: it's time to change our mind. Curr Neuropharmacol. 2017;15(6):926-35.
  • Selkoe DJ, Hardy J. The amyloid hypothesis of Alzheimer's disease at 25 years. EMBO Mol Med. 2016;8(6):595-608.
  • Selkoe DJ. Alzheimer's disease is a synaptic failure. Science. 2002;298(5594):789-91.
  • Kuperstein I, Broersen K, Benilova I, et al. Neurotoxicity of Alzheimer's disease Aβ peptides is induced by small changes in the Aβ42 to Aβ40 ratio. EMBO J. 2010;29(19):3408-20.
  • Kumar‐Singh S, Theuns J, Van Broeck B, et al. Mean age‐of‐onset of familial alzheimer disease caused by presenilin mutations correlates with both increased Aβ42 and decreased Aβ40. Hum Mutat. 2006;27(7):686-95.
  • Kim J, Onstead L, Randle S, et al. Aβ40 inhibits amyloid deposition in vivo. J Neurosci. 2007;27(3):627-33.
  • Yoshiike Y, Chui D-H, Akagi T, et al. Specific compositions of amyloid-β peptides as the determinant of toxic β-aggregation. J Biol Chem. 2003;278(26):23648-55.
  • Frost D, Gorman PM, Yip CM, et al. Co‐incorporation of Aβ40 and Aβ42 to form mixed pre‐fibrillar aggregates. Eur J Biochem. 2003;270(4):654-63.
  • Chasseigneaux S, Allinquant B. Functions of Aβ, sAPPα and sAPPβ: similarities and differences. J Neurochem.2012;120:99-108.
  • Cai Z, Zhao Y, Zhao B. Roles of glycogen synthase kinase 3 in Alzheimer's disease. Curr Alzheimer Res. 2012;9(7):864-79.
  • Uemura K, Kuzuya A, Shimozono Y, et al. GSK3β activity modifies the localization and function of presenilin 1. J Biol Chem. 2007;282(21):15823-32.
  • Carvajal FJ, Inestrosa NC. Interactions of AChE with Aβ aggregates in Alzheimer’s brain: therapeutic relevance of IDN 5706. Front Mol Neurosci. 2011;4:19.
  • Dinamarca MC, Sagal JP, Quintanilla RA, et al. Amyloid-β-Acetylcholinesterase complexes potentiate neurodegenerative changes induced by the Aβ peptide. Implications for the pathogenesis of Alzheimer's disease. Mol Neurodegener. 2010;5:1-15.
  • Götz J, Eckert A, Matamales M, et al. Modes of Aβ toxicity in Alzheimer’s disease. Cell Mol Life Sci. 2011;68:3359-75.
  • Wischik C, Edwards P, Lai R, Roth M, Harrington C. Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines. PNAS. 1996;93(20):11213-8.
  • Mori T, Koyama N, Segawa T, Maeda M, Maruyama N, Kinoshita N, et al. Methylene blue modulates β-secretase, reverses cerebral amyloidosis, and improves cognition in transgenic mice. J Biol Chem. 2014;289(44):30303-17.

Tiyoninin N2a-APPSwe Hücrelerinde Amiloid Prekürsör Protein İşlenmesi Üzerindeki Rolü

Year 2025, Volume: 12 Issue: 2, 161 - 167, 28.08.2025
https://doi.org/10.47572/muskutd.1698250

Abstract

Amiloid prekürsör proteininin (APP) sekretazlar tarafından kırılmasıyla oluşan amiloid-β (Aβ) peptitlerinin anormal birikimiyle karakterize Alzheimer hastalığı, bilişsel bozukluk ile ilişkilidir. Kanıtlar, kolinerjik aktivitenin, APP işlenmesinin regülasyonunu değiştirebileceğini göstermiştir. Bu araştırmanın amacı, fenotiyazin yapılı bir kolinesteraz inhibitörü olan tiyoninin (TH), APP'nin İsveç mutantını aşırı eksprese eden N2a fare nöroblastoma hücrelerinde (N2a/APPSwe hücreleri) APP işlenmesi üzerindeki etkisini değerlendirmektir. Bu amaçla, tiyoninin Aβ40/42 peptitleri, çözünür APPα (sAPPα), hücre içi APP, β-bölge APP-kesim enzimi 1 (BACE-1), presenilin-1 (PS1) ve glikojen sentaz kinaz 3 beta (GSK3β) düzeylerini nasıl etkilediğini araştırdık. N2a/APPSwe hücreleri 24 saat boyunca 1.25-10 μM tiyonin ile muamele edildikten sonra, ELISA yöntemi kullanılarak Aβ42, Aβ40 ve sAPPα düzeyleri değerlendirilmiştir. Ayrıca, tiyoninin hücre içi APP, PS1 ve GSK3β düzeyleri üzerindeki etkileri, Western blot yöntemi ile analiz edilmiştir. Bulgularımız, tiyoninin N2a/APPSwe hücrelerinde Aβ peptitler ve APP düzeylerini önemli ölçüde azalttığını göstermiştir. Tiyoninin APP işlenmesi üzerindeki etkilerini değerlendirdiğimizde, BACE-1 düzeyinde önemli bir değişiklik gözlenmemiştir. Ancak PS1 ve GSK3β düzeylerinde istatistiksel olarak anlamlı bir azalma gözlenmiştir. Sonuçlar, tiyoninin amiloid patolojisini önleme potansiyeline ilişkin önemli bir etki mekanizması sunmaktadır.

Project Number

TSA-2019-17288

References

  • Toodayan N. Professor Alois Alzheimer (1864–1915): lest we forget. J Clin Neurosci. 2016;31:47-55.
  • Janoutová J, Kovalová M, Machaczka O, et al. Risk factors for Alzheimer’s disease: an epidemiological study. Curr Alzheimer Res. 2021;18(5):372-9.
  • Dos Santos Picanco LC, Ozela PF, de Fatima de Brito Brito M, et al. Alzheimer's disease: a review from the pathophysiology to diagnosis, new perspectives for pharmacological treatment. Curr Med Chem. 2018;25(26):3141-59.
  • Kang J, Lemaire H-G, Unterbeck A, et al. The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature. 1987;325(6106):733-6.
  • Haass C, Selkoe DJ. Cellular processing of beta-amyloid precursor protein and the genesis of amyloid beta-peptide. Cell. 1993;75(6):1039-42.
  • Chen J, Chen J-S, Li S, Zhang F, et al. Amyloid precursor protein: a regulatory hub in Alzheimer's disease. Aging Dis. 2024;15(1):201.
  • Steiner H, Fukumori A, Tagami S, et al. Making the final cut: pathogenic amyloid-β peptide generation by γ-secretase. Cell Stress. 2018;2(11):292.
  • Ittner LM, Götz J. Amyloid-β and tau—a toxic pas de deux in Alzheimer's disease. Nat Rev Neurosci. 2011;12(2):67-72.
  • Green FJ. The Sigma-Aldrich handbook of stains, dyes, and indicators. 1990.
  • Wainwright M, Phoenix D, Laycock S, et al. Photobactericidal activity of phenothiazinium dyes against methicillin-resistant strains of Staphylococcus aureus. FEMS Microbiol Lett. 1998;160(2):177-81.
  • Harris F, Chatfield L, Phoenix D. Phenothiazinium based photosensitisers-photodynamic agents with a multiplicity of cellular targets and clinical applications. Curr Drug Targets. 2005;6(5):615-27.
  • Gualdesi MS, Vara J, Aiassa V, Igarzabal CA, Ortiz CS. Thionine in the design of new photosensitizers: Bromination and vehiculization in polymeric nanoparticles. J Mol Liq. 2020;310:113247.
  • Yuksel M, Biberoglu K, Onder S, et al. Effects of phenothiazine-structured compounds on APP processing in Alzheimer's disease cellular model. Biochimie. 2017;138:82-9.
  • Onder S, Biberoglu K, Yuksel M, et al. Toluidine blue O attenuates tau phosphorylation in N2a-APPSwe cells. Chem Biol Interact. 2022;366:110126.
  • Biberoglu K, Tek MY, Ghasemi ST, et al. Toluidine blue O is a potent inhibitor of human cholinesterases. Arch Biochem Biophys. 2016;604:57-62.
  • Onder S, Biberoglu K, Yuksel M, et al. Fenotı̇yazı̇n Yapısındakı̇ Kolı̇nesteraz İnhı̇bı̇törlerı̇nı̇n Amı̇loı̇d Prekürsör Proteı̇n Metabolı̇zması Ve Tau Üzerı̇ndekı̇ Etkı̇ Mekanı̇zmalarının Araştırılması, Hacettepe Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi. 2019.
  • Loetscher H, Deuschle U, Brockhaus M, et al. Presenilins are processed by caspase-type proteases. J Biol Chem. 1997;272(33):20655-9.
  • Lauretti E, Dincer O, Praticò D. Glycogen synthase kinase-3 signaling in Alzheimer's disease. Biochim Biophys Acta. 2020;1867(5):118664.
  • Kurkinen M, Fułek M, Fułek K, et al. The amyloid cascade hypothesis in Alzheimer’s disease: should we change our thinking? Biomolecules. 2023;13(3):453.
  • Majdi A, Sadigh-Eteghad S, Rahigh Aghsan, et al. Amyloid-β, tau, and the cholinergic system in Alzheimer’s disease: Seeking direction in a tangle of clues. Rev Neurosci. 2020;31(4):391-413.
  • Haass C, Selkoe DJ. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid β-peptide. Nat Rev Mol Cell Biol. 2007;8(2):101-12.
  • Ricciarelli R, Fedele E. The amyloid cascade hypothesis in Alzheimer's disease: it's time to change our mind. Curr Neuropharmacol. 2017;15(6):926-35.
  • Selkoe DJ, Hardy J. The amyloid hypothesis of Alzheimer's disease at 25 years. EMBO Mol Med. 2016;8(6):595-608.
  • Selkoe DJ. Alzheimer's disease is a synaptic failure. Science. 2002;298(5594):789-91.
  • Kuperstein I, Broersen K, Benilova I, et al. Neurotoxicity of Alzheimer's disease Aβ peptides is induced by small changes in the Aβ42 to Aβ40 ratio. EMBO J. 2010;29(19):3408-20.
  • Kumar‐Singh S, Theuns J, Van Broeck B, et al. Mean age‐of‐onset of familial alzheimer disease caused by presenilin mutations correlates with both increased Aβ42 and decreased Aβ40. Hum Mutat. 2006;27(7):686-95.
  • Kim J, Onstead L, Randle S, et al. Aβ40 inhibits amyloid deposition in vivo. J Neurosci. 2007;27(3):627-33.
  • Yoshiike Y, Chui D-H, Akagi T, et al. Specific compositions of amyloid-β peptides as the determinant of toxic β-aggregation. J Biol Chem. 2003;278(26):23648-55.
  • Frost D, Gorman PM, Yip CM, et al. Co‐incorporation of Aβ40 and Aβ42 to form mixed pre‐fibrillar aggregates. Eur J Biochem. 2003;270(4):654-63.
  • Chasseigneaux S, Allinquant B. Functions of Aβ, sAPPα and sAPPβ: similarities and differences. J Neurochem.2012;120:99-108.
  • Cai Z, Zhao Y, Zhao B. Roles of glycogen synthase kinase 3 in Alzheimer's disease. Curr Alzheimer Res. 2012;9(7):864-79.
  • Uemura K, Kuzuya A, Shimozono Y, et al. GSK3β activity modifies the localization and function of presenilin 1. J Biol Chem. 2007;282(21):15823-32.
  • Carvajal FJ, Inestrosa NC. Interactions of AChE with Aβ aggregates in Alzheimer’s brain: therapeutic relevance of IDN 5706. Front Mol Neurosci. 2011;4:19.
  • Dinamarca MC, Sagal JP, Quintanilla RA, et al. Amyloid-β-Acetylcholinesterase complexes potentiate neurodegenerative changes induced by the Aβ peptide. Implications for the pathogenesis of Alzheimer's disease. Mol Neurodegener. 2010;5:1-15.
  • Götz J, Eckert A, Matamales M, et al. Modes of Aβ toxicity in Alzheimer’s disease. Cell Mol Life Sci. 2011;68:3359-75.
  • Wischik C, Edwards P, Lai R, Roth M, Harrington C. Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines. PNAS. 1996;93(20):11213-8.
  • Mori T, Koyama N, Segawa T, Maeda M, Maruyama N, Kinoshita N, et al. Methylene blue modulates β-secretase, reverses cerebral amyloidosis, and improves cognition in transgenic mice. J Biol Chem. 2014;289(44):30303-17.
There are 37 citations in total.

Details

Primary Language English
Subjects Clinical Sciences (Other)
Journal Section Original Article
Authors

Seda Onder 0000-0002-0392-7077

Kevser Bıberoglu 0000-0001-9285-0819

Özden Tacal 0000-0002-2772-5226

Project Number TSA-2019-17288
Publication Date August 28, 2025
Submission Date May 13, 2025
Acceptance Date July 31, 2025
Published in Issue Year 2025 Volume: 12 Issue: 2

Cite

APA Onder, S., Bıberoglu, K., & Tacal, Ö. (2025). The Role of Thionine on Amyloid Precursor Protein Processing in N2a-APPSwe Cells. Muğla Sıtkı Koçman Üniversitesi Tıp Dergisi, 12(2), 161-167. https://doi.org/10.47572/muskutd.1698250
AMA Onder S, Bıberoglu K, Tacal Ö. The Role of Thionine on Amyloid Precursor Protein Processing in N2a-APPSwe Cells. MMJ. August 2025;12(2):161-167. doi:10.47572/muskutd.1698250
Chicago Onder, Seda, Kevser Bıberoglu, and Özden Tacal. “The Role of Thionine on Amyloid Precursor Protein Processing in N2a-APPSwe Cells”. Muğla Sıtkı Koçman Üniversitesi Tıp Dergisi 12, no. 2 (August 2025): 161-67. https://doi.org/10.47572/muskutd.1698250.
EndNote Onder S, Bıberoglu K, Tacal Ö (August 1, 2025) The Role of Thionine on Amyloid Precursor Protein Processing in N2a-APPSwe Cells. Muğla Sıtkı Koçman Üniversitesi Tıp Dergisi 12 2 161–167.
IEEE S. Onder, K. Bıberoglu, and Ö. Tacal, “The Role of Thionine on Amyloid Precursor Protein Processing in N2a-APPSwe Cells”, MMJ, vol. 12, no. 2, pp. 161–167, 2025, doi: 10.47572/muskutd.1698250.
ISNAD Onder, Seda et al. “The Role of Thionine on Amyloid Precursor Protein Processing in N2a-APPSwe Cells”. Muğla Sıtkı Koçman Üniversitesi Tıp Dergisi 12/2 (August2025), 161-167. https://doi.org/10.47572/muskutd.1698250.
JAMA Onder S, Bıberoglu K, Tacal Ö. The Role of Thionine on Amyloid Precursor Protein Processing in N2a-APPSwe Cells. MMJ. 2025;12:161–167.
MLA Onder, Seda et al. “The Role of Thionine on Amyloid Precursor Protein Processing in N2a-APPSwe Cells”. Muğla Sıtkı Koçman Üniversitesi Tıp Dergisi, vol. 12, no. 2, 2025, pp. 161-7, doi:10.47572/muskutd.1698250.
Vancouver Onder S, Bıberoglu K, Tacal Ö. The Role of Thionine on Amyloid Precursor Protein Processing in N2a-APPSwe Cells. MMJ. 2025;12(2):161-7.