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

Abstract

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.

Keywords

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

Supporting Institution

PAMUKKALE ÜNİVERSİTESİ BİLİMSEL ARAŞTIRMA PROJELERİ KOORDİNATÖRLÜĞÜ , TÜBİTAK

Project Number

PAUBAP: 2020SABE17, TÜBİTAK 1002: 120Z825

Ethical Statement

Bu çalışma PAMUKKALE ÜNİVERSİTESİ BİLİMSEL ARAŞTIRMA PROJELERİ KOORDİNATÖRLÜĞÜ ve TÜBİTAK (1002) tarafından desteklenmiştir.

Thanks

Araştırma personeli PAMUKKALE ÜNİVERSİTESİ BİLİMSEL ARAŞTIRMA PROJELERİ KOORDİNATÖRLÜĞÜ ve TÜBİTAK'a destekleri için teşekkür eder.

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

Abstract

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.

Keywords

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

Project Number

PAUBAP: 2020SABE17, TÜBİTAK 1002: 120Z825

Thanks

The research staff would like to thank the PAMUKKALE UNIVERSITY COORDINATORSHIP OF SCIENTIFIC RESEARCH PROJECTS and TÜBİTAK for their support (PAUBAP: 2020SABE17, TÜBİTAK 1002: 120Z825).

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