S-sülfosistein ile tetiklenen hücre ölümünde kortikal ve hipokampal nöronlarda kaspaz-3 bağımlı ve bağımsız apoptotik süreçlerin in vitro incelenmesi

Öz

Amaç: Kükürt içeren amino asit metabolizmasının toksik bir yan ürünü olan S-sülfosistein (SSC), molibden kofaktör eksikliği (MoCD) ve izole sülfit oksidaz eksikliği (iSOD) gibi durumlarda birikir ve ciddi nörodejenerasyona yol açar. SSC’nin nörotoksisitesi kanıtlanmış olsa da özellikle farklı beyin bölgelerinde tetiklediği apoptoz mekanizmaları tam olarak açıklığa kavuşmamıştır. Bu çalışma, SSC’nin hipokampal HT-22 ve primer kortikal nöronlarda oluşturduğu bölgeye özgü apoptoz yollarını, kaspaz-bağımlı ve bağımsız mekanizmalar açısından araştırmayı amaçlamaktadır. Gereç ve yöntem: Nöronal hücreler artan dozlarda SSC ile muamele edilmiştir. Hücre canlılığı, glutatyon (GSH) düzeyleri ve apoptoza ilişkin proteinler (AIF, kalpain, sitokrom c, kaspaz-3) sırasıyla CCK-8 testi, enzimatik GSH testi ve ELISA yöntemi ile analiz edilmiştir. AIF, kalpain ve sitokrom c’yi hedef alan spesifik inhibitörlerin koruyucu etkileri de değerlendirilmiştir. Bulgular: SSC, her iki hücre modelinde de canlılığı azaltmış; yarı maksimal letal doz (LD50) değerleri HT-22 hücrelerinde 150 μM, kortikal nöronlarda ise 155 μM olarak belirlenmiştir. SSC, her iki modelde AIF ve kalpain düzeylerini artırmış, ancak sitokrom c ve kaspaz-3 düzeyleri yalnızca kortikal nöronlarda anlamlı olarak yükselmiştir. GSH düzeyleri 2–8 saat arasında artmış, 16. saate gelindiğinde azalmıştır. AIF, kalpain ve sitokrom c inhibitörleri hücre canlılığını kısmen geri kazandırmış; kombinasyon tedavisi en güçlü koruyucu etkiyi göstermiştir. Sonuç: SSC, bölgeye özgü şekilde hem kaspaz-bağımsız hem de kaspaz-bağımlı apoptozu tetiklemektedir: HT-22 hücrelerinde ağırlıklı olarak AIF ve kalpain mekanizmaları devreye girerken, kortikal nöronlarda buna ek olarak sitokrom c ve kaspaz-3 de aktive olmaktadır. Bu bulgular, bölgesel moleküler hassasiyetleri ortaya koymakta ve sülfite bağlı nörodejeneratif hastalıklar için potansiyel tedavi hedefleri sunmaktadır.

Anahtar Kelimeler

• S-sülfosistein
• apoptoz
• primer kortikal nöronlar
• oksidatif stres
• sülfit oksidaz eksikliği

In vitro investigation of caspase-3 dependent and independent apoptotic processes in cortical and hippocampal neurons triggered by S-sulfocysteine-induced cell death

Öz

Purpose: S-sulfocysteine (SSC), a toxic byproduct of sulfur-containing amino acid metabolism, accumulates in conditions such as molybdenum cofactor deficiency (MoCD) and isolated sulfite oxidase deficiency (iSOD), leading to severe neurodegeneration. Despite evidence of SSC's neurotoxicity, the apoptotic mechanisms it triggers remain unclear, particularly in different brain regions. This study aimed to investigate the region-specific apoptotic pathways induced by SSC in hippocampal HT-22 and primary cortical neurons, focusing on caspase-dependent and -independent mechanisms. Materials and methods: Neuronal cells were treated with increasing doses of SSC, and cell viability, glutathione (GSH) levels, and apoptosis-related proteins (AIF, calpain, cytochrome c, caspase-3) were assessed using CCK-8 assay, GSH enzymatic assay, and ELISA. The protective effects of specific inhibitors targeting AIF, calpain, and cytochrome c were also evaluated. Results: SSC reduced cell viability in both neuronal types with half-maximal lethal dose (LD50) values of 150 μM (HT-22) and 155 μM (cortical neurons). In both models, SSC elevated AIF and calpain levels, whereas cytochrome c and caspase-3 were significantly increased only in cortical neurons. GSH levels initially rose at 2–8 hours and declined by 16 hours. Inhibitors of AIF, calpain, and cytochrome c partially restored viability, with combined administration offering the most robust protection. Conclusion: SSC induces both caspase-independent and caspase-dependent apoptosis in a region-specific manner: HT-22 cells predominantly activate AIF and calpain, while cortical neurons engage additional cytochrome c and caspase-3 pathways. These findings suggest distinct molecular vulnerabilities and offer potential targets for therapeutic intervention in sulfite-related neurodegenerative diseases.

Anahtar Kelimeler

• S-sulfocysteine
• apoptosis
• primary cortical neurons
• oxidative stress
• sulfite oxidase deficiency

Kaynakça

1. Sbodio JI, Snyder SH, Paul BD. Regulators of the transsulfuration pathway. Br J Pharmacol. 2019;176(4):583-593. doi: 10.1111/bph.14446
2. Sigel A, Sigel H, Sigel RKO. Interrelations between Essential Metal Ions and Human Diseases. Springer. 2013:573. https://link.springer.com/book/10.1007/978-94-007-7500-8
3. Schwarz G. Molybdenum cofactor and human disease. Curr Opin Chem Biol. 2016;31:179-187. doi:10.1016/j.cbpa.2016.03.016
4. Claerhout H, Witters P, Regal L, et al. Isolated sulfite oxidase deficiency. J Inherit Metab Dis. 2018;41(1):101-108. doi:10.1007/s10545-017-0089-4
5. Kishikawa M, Sass JO, Sakura N, et al. The peak height ratio of S sulfonated transthyretin. Biochim Biophys Acta. 2002;1588(2):135-138. doi:10.1016/s0925-4439(02)00156-4.
6. Kumar A, Dejanovic B, Hetsch F, et al. S-sulfocysteine/NMDA receptor–dependent signaling underlies neurodegeneration in molybdenum cofactor deficiency. J Clin Invest. 2017;127(12):4365-4378. doi:10.1172/JCI93707
7. Grings M, Moura AP, Parmeggiani B, et al. Higher susceptibility of cerebral cortex and striatum to sulfite neurotoxicity. Biochim Biophys Acta. 2016;1862(11):2063-2074. doi:10.1016/j.bbamem.2016.09.004
8. Zhang X, Chen J, Graham SH, et al. Intranuclear localization of AIF and large-scale DNA fragmentation after TBI. J Neurochem. 2002;82(1):181-191. doi:10.1046/j.1471-4159.2002.00975.x

9. Candé C, Cecconi F, Dessen P, Kroemer G. AIF: key to the conserved caspase-independent pathways of cell death? J Cell Sci. 2002;115(Pt 24):4727-4734. doi:10.1242/jcs.00210
10. Jiang X, Wang X. Cytochrome C–mediated apoptosis. Annu Rev Biochem. 2004;73:87-106. doi:10.1146/annurev.biochem.73.011303.073706
11. Verma M, Kaganovich D. Excitotoxicity, calcium and mitochondria: a triad in synaptic neurodegeneration. Transl Neurodegener. 2022;11:3. doi:10.1186/s40035-021-00278-7
12. Wu W, Gong X, Qin ZH, et al. Molecular mechanisms of excitotoxicity and their relevance to the pathogenesis of neurodegenerative diseases—an update. Acta Pharmacol Sin. 2025;46(1):1-15. doi:10.1038/s41401-025-01576-w
13. Yamada J, Jinno S. Differential susceptibility of cortical and hippocampal interneurons to NMDA-induced excitotoxicity. Neuroscience. 2021;459:88-101. doi:10.1016/j.neuroscience.2021.01.028
14. Gouix E, Moulis E, Bertrand L, et al. Region-specific susceptibility of hippocampal subfields to NMDA receptor-mediated excitotoxicity. J Neurochem. 2023;166(5):633-647. doi:10.1111/jnc.15776
15. Zhang Y, Bhavnani BR. Glutamate-induced apoptosis in neuronal cells. BMC Neurosci. 2006;7:49. doi:10.1186/1471-2202-7-49
16. Olney JW. Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate. Science. 1969;164(3880):719-721. doi:10.1126/science.164.3880.719.
17. Dong X, Wang Y, Qin ZH. Molecular mechanisms of excitotoxicity. Acta Pharmacol Sin. 2009;30(4):379-387. doi:10.1038/aps.2009.22
18. Verma M, Lizama BN, Chu CT. Excitotoxicity, calcium and mitochondria. Transl Neurodegener. 2022;11(1):3. doi:10.1186/s40035-021-00277-0
19. Abdellatif M, Kroemer G. Exercise induced sudden cardiac death is caused by mitochondrio nuclear translocation of AIF. Cell Death Dis. 2021;12(4):383. doi:10.1038/s41419-021-03677-w
20. Cande C, Vahsen N, Garrido C, Kroemer G. AIF: caspase-independent after all. Cell Death Differ. 2004;11(6):591-595. doi:10.1038/sj.cdd.4401380
21. Fukui M, Song JH, Choi J, et al. Mechanism of glutamate-induced neurotoxicity in HT 22 cells. Eur J Pharmacol. 2009;617(1-3):1-11. doi:10.1016/j.ejphar.2009.06.059
22. Tobaben S, Grohm J, Seiler A, et al. Bid-mediated mitochondrial damage in glutamate-induced oxidative stress and AIF-dependent death in HT 22. Cell Death Differ. 2011;18(2):282-292. doi:10.1038/cdd.2010.92
23. Yu SW, Wang H, Dawson TM, Dawson VL. PARP 1 and AIF in neurotoxicity. Neurobiol Dis. 2003;14(3):303-317. doi:10.1016/S0969-9961(03)00015-7
24. Hill CA, Fitch RH. Sex differences in neonatal hypoxia-ischemia mechanisms and outcomes. Neurol Res Int. 2012;2012:867531. doi:10.1155/2012/867531
25. Andrabi SA, Kim NS, Yu SW, et al. Poly(ADP ribose) polymer is a death signal. Proc Natl Acad Sci U S A. 2006;103(48):18308-18313. doi:10.1073/pnas.0606526103
26. Kritis AA, Stamoula EG, Paniskaki KA, Vavilis TD. Researching glutamate-induced cytotoxicity in different cell lines. Front Cell Neurosci. 2015;9:91. doi:10.3389/fncel.2015.00091
27. Angelova PR, Abramov AY. Interplay of mitochondrial calcium signalling and reactive oxygen species production in the brain. Biochem Soc Trans. 2024;52(4):1939-1946. doi:10.1042/BST20240261
28. Szydlowska K, Tymianski M. Calcium, ischemia and excitotoxicity. Cell Calcium. 2022;102:102508. doi:10.1016/j.ceca.2021.102508
29. Leyen K, Siddiq A, Ratan RR, Lo EH. Proteasome inhibition protects HT22 cells from oxidative glutamate toxicity. J Neurochem. 2005;92(4):824-830. doi:10.1111/j.1471-4159.2004.02915.x
30. Susin SA, Zamzami N, Castedo M, et al. Bcl 2 inhibits mitochondrial release of an apoptogenic protease. J Exp Med. 1996;184(4):1331-1341. doi:10.1084/jem.184.4.1331
31. Ding L, Li J, Li W, et al. p53 and ROS mediated AIF pathway involved in TGEV induced apoptosis. J Vet Med Sci. 2018;80(11):1775-1781. doi:10.1292/jvms.18-0104
32. Yoshikawa Y, Hagihara H, Ohga Y, et al. Calpain inhibitor 1 protects rat heart from ischemia reperfusion injury. Am J Physiol Heart Circ Physiol. 2005;288(4):H1690-H1698. doi:10.1152/ajpheart.00666.2004
33. Wang X, Zhu S, Pei Z, et al. Inhibitors of cytochrome c release with therapeutic potential for Huntington’s disease. J Neurosci. 2008;28(38):9473-9485. doi:10.1523/JNEUROSCI.1867-08.2008
34. Fossati S, Todd K, Sotolongo K, et al. Differential contribution of isoaspartate post translational modifications to amyloid-β toxicity. Biochem J. 2013;456(3):347-360. doi:10.1042/BJ20130652