The Effect of Ca-074 (Cathepsın B Inhibitor) on Necrotic and Apoptotic Neuronal Cell Death in Model of Cerebral Ischemia

Year 2023, Volume: 45 Issue: 5, 781 - 790, 27.09.2023

Emre Özkara , Ramazan Durmaz , Zühtü Özbek , Hilmi Özden , Güngör Kanbak , Kubilay Uzuner

https://doi.org/10.20515/otd.1343599

Abstract

Lysosomes and cathepsins, the most common hydrolytic enzymes in lysosomes, are available in the different models of cell death as necrosis and apoptosis. This study investigated the effect of cathepsin B-selective inhibitor CA-074 on apoptotic and necrotic neuronal cell death. Focal cerebral ischemia which has been formed by occlusion of the three-vessel consisting permanent middle cerebral artery occlusion and temporary bilateral common carotid artery occlusion for 60 minutes was selected as ischemia model. Two sets of rats were used in this study. The rats in the first set were used formeasurement of sulfhydryl groups in the lysosomal membrane, lysosomal integrity, cathepsins B and L activities and caspase-3 activity. The rats in the second set were used as histological study including "hematoxylin and eosin" for the detection of necrotic neuronal deathand "TUNEL" staining for the detection of apoptotic neuronal death. 4 mg/kg CA-074 was administered intravenouslyin the treatment group. CA-074 has substantially reduced levels of cathepsins B and L compared to ischemia and solvent groups (respectively, p<0.05 and p<0.01). Similarly, CA-074 has reduced increase in caspase-3 activity compared to ischemia and solvent groups (p<0.05). While the number of eosinophilic (necrotic) and apoptotic neurons has highly increased in post-ischemic cerebral tissue in middle cerebral artery feeding area (p<0.001), CA-074 could only reduce significantly the number of apoptotic neurons (p<0.05). CA-074 has reduced apoptotic neuronal death by inhibiting caspase and cathepsin activity. It may be useful that CA074 is used with other therapeutic drugs in stroke patients.

Keywords

ischemia, cerebral, rat model, CA074

Katepsin B İnhibitörü olan CA074'ün Serebral İskemi Modelinde Apoptoz ve Hücre Ölümü Üzerine Etkisi

Abstract

Lizozomlarda en yaygın hidrolitik enzimlerden olan lizozomlar ve katepsinler, nekroz ve apoptoz olarak farklı hücre ölümü modellerinde görev almaktadır. Bu çalışma, katepsin B-seçici inhibitörü olan CA-074'ün apoptotik ve nekrotik nöronal hücre ölümü üzerindeki etkisini araştırdı. Bu çalışmda İskemi modeli olarak kalıcı orta serebral arterin tıkanıklığı ve geçici bilateral ana karotid arter tıkanıklığından oluşan üç damarın 60 dakika süreyle oklüzyonu ile oluşturulan fokal serebral iskemi modeli seçilmiştir. Çalışmada iki set sıçan kullanıldı. Birinci setteki ratlar lizozomal membrandaki sülfhidril gruplarının, lizozomal bütünlüğün, katepsin B ve L aktivitelerinin ve kaspaz-3 aktivitesinin ölçülmesinde kullanıldı. İkinci setteki sıçanlar, nekrotik nöronal ölümün saptanması için "hematoksilen ve eozin" ve apoptotik nöronal ölümün saptanması için "TUNEL" boyamasını içeren histolojik çalışma olarak kullanıldı. Tedavi grubuna 4 mg/kg CA-074 intravenöz olarak uygulandı. CA-074, iskemi ve solvent gruplarına kıyasla önemli ölçüde azalmış katepsin B ve L seviyelerine sahiptir (sırasıyla, p<0.05 ve p<0.01). Benzer şekilde CA-074 kaspaz-3 aktivitesindeki artışı iskemi ve solvent gruplarına göre azaltmıştır (p<0.05). Orta serebral arter beslenme alanındaki iskemik sonrası serebral dokuda eozinofilik (nekrotik) ve apoptotik nöronların sayısı oldukça artarken (p<0.001), CA-074 sadece apoptotik nöronların sayısını önemli ölçüde azaltabildi (p<0.05). CA-074, kaspaz ve katepsin aktivitesini inhibe ederek apoptotik nöronal ölümü azaltmıştır. CA074'ün inme hastalarında diğer terapötik ilaçlarla birlikte kullanılması faydalı olabilir.

Keywords

iskemi, beyin, rat modeli, CA074

Supporting Institution

Eskişehir Osmangazi Üniversitesi

References

• 1. Brojatsch J, Lima H, Kar AK, Jacobson LS, Muehlbauer SM, Chandran K, Diaz-Griffero F. A proteolytic cascade controls lysosome rupture and necrotic cell death mediated by lysosome-destabilizing adjuvants. PLoS One. 2014;9(6):1-9.
• 2. Repnik U, Stoka V, Turk V, Turk B. Lysosomes and lysosomal cathepsins in cell death. Biochim Biophys Acta. 2012; 1824:22–33.
• 3. Guicciardi ME, Gores GJ. Complete lysosomal disruption: a route to necrosis, not to the inflammasome. Cell Cycle. 2013; 12(13):1995.
• 4. Pope, A. and Nixon, R.A.Proteases of human brain. Neurochem. Res. 1994; 9:291–323.
• 5. Benchoua A, Braudeau J, Reis A, Couriaud C, Onteniente B. Activation of proinflammatory caspases by cathepsin B in focal cerebral ischemia. J Cereb Blood Flow Metab. 2004; 24(11):1272-9.
• 6. Yamashima T, Kohda Y, Tsuchiya K, Ueno T, Yamashita J, Yoshioka T, Kominami E. Inhibition of ischaemic hippocampal neuronal death in primates with cathepsin B inhibitor CA-074: a novel strategy for neuroprotection based on 'calpain-cathepsin hypothesis'. Eur J Neurosci. 1998; 10(5); 1723-33.
• 7. Yanamoto H, Nagata I, Niitsu Y, Xue JH, Zhang Z, Kikuchi H. Evaluation of MCAO stroke models in normotensive rats: standardized neocortical infarction by the 3VO technique. Exp Neurol. 2003; 182(2):261-74.
• 8. Yamashima T. Ca2+-dependent proteases in ischemic neuronal death: a conserved 'calpain-cathepsin cascade' from nematodes to primates. Cell Calcium. 2004; 36(3-4):285-93.
• 9. De Duve C. In: Hayashi T, ed. Subcellular Particles New York: Ronald Press Co., 1952:128–59.
• 10. Reinheckel T. On the road to inflammation: linking lysosome disruption, lysosomal protease release and necrotic death of immune cells. Cell Cycle. 2013; 12(13):1994.
• 11. Festjens N, Vanden Berghe T, and Vandenabeele P. Necrosis, a wellorchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response. Biochim Biophys Acta.2006; 1757: 1371–1387.
• 12. Fink SL, Cookson BT. Caspase-1-dependent pore formation during pyroptosis leads to osmotic lysis of infected host macrophages. Cell Micro. 2006; 8: 1812–1825.
• 13. Brennan MA, Cookson BT. Salmonella induces macrophage death by caspase-1-dependent necrosis. Mol Micro. 2006; 38: 31–40.
• 14. Keller M, Ruegg A, Werner S, Beer HD. Active caspase-1 is a regulator of unconventional protein secretion. Cell. 2008; 132: 818–831.
• 15. Berghe TV, Vanlangenakker N, Parthoens E, Deckers W, Devos M, et al. Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features. Cell Death Differ. 2010; 17: 922–930.
• 16. Christofferson DE, Yuan JY. Necroptosis as an alternative form of programmed cell death. Curr Opin Cell Biol. 2010; 22: 263–268
• 17. Lima H Jr, Jacobson LS, Goldberg MF, Chandran K, Diaz-Griffero F, et al. Role of lysosome rupture in controlling Nlrp3 signaling and necrotic cell death. Cell Cycle. 2013; 12: 1868–1878.
• 18. Guicciardi ME, Gores GJ. Complete lysosomal disruption: a route to necrosis, not to the inflammasome. Cell Cycle. 2013; 12: 1995.
• 19. Turk B, Turk D, Turk V. Lysosomal cysteine proteases: more than scavengers. Biochim Biophys Acta. 2000; 1477(1-2):98-111.
• 20. Lecaille F, Kaleta J, Brömme D. Human and parasitic papain-like cysteine proteases: their role in physiology and pathology and recent developments in inhibitor design. Chem Rev. 2002; 102(12):4459-88.
• 21. Nitatori T, Sato N, Kominami E, Uchiyama Y. Participation of cathepsins B, H, and L in perikaryal condensation of CA1 pyramidal neurons undergoing apoptosis after brief ischemia. Adv Exp Med Biol. 1996; 389:177-85.
• 22. Hill IE, Preston E, Monette R, MacManus JP. A comparison of cathepsin B processing and distribution during neuronal death in rats following global ischemia or decapitation necrosis. Brain Res. 1997; 751(2):206-16.
• 23. Seyfried D, Han Y, Zheng Z, Day N, Moin K, Rempel S, Sloane B, Chopp M. Cathepsin B and middle cerebral artery occlusion in the rat. J Neurosurg. 1997;87(5):716-23.
• 24. Kohda Y, Yamashima T, Sakuda K, Yamashita J, Ueno T, Kominami E, Yoshioka T. Dynamic changes of cathepsins B and L expression in the monkey hippocampus after transient ischemia. Biochem Biophys Res Commun. 1996; 228(2):616-22.
• 25. Towatari T, Nikawa T, Murata M, Yokoo C, Tamai M, Hanada K, Katunuma N. Novel epoxysuccinyl peptides. A selective inhibitor of cathepsin B, in vivo. FEBS Lett. 1991;280(2):311-5.
• 26. Blomgren K, Zhu C, Wang X, Karlsson JO, Leverin AL, Bahr BA, Mallard C, Hagberg H. Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of "pathological apoptosis"? J Biol Chem. 2001; 276(13):10191-8.
• 27. Rami A. Ischemic neuronal death in the rat hippocampus: the calpain-calpastatin-caspase hypothesis. Neurobiol Dis. 2003; 13(2):75-88.
• 28. Tardy C, Codogno P, Autefage H, Levade T, Andrieu-Abadie N. Lysosomes and lysosomal proteins in cancer cell death (new players of an old struggle). Biochim Biophys Acta. 2006;1765(2):101-25.
• 29. Zhu C, Qiu L, Wang X, Hallin U, Candé C, Kroemer G, Hagberg H, Blomgren K. Involvement of apoptosis-inducing factor in neuronal death after hypoxia-ischemia in the neonatal rat brain. J Neurochem. 2003; 86(2):306-17.
• 30. Baud O, Li J, Zhang Y, Neve RL, Volpe JJ, Rosenberg PA. Nitric oxide-induced cell death in developing oligodendrocytes is associated with mitochondrial dysfunction and apoptosis-inducing factor translocation. Eur J Neurosci. 2004; 20(7):1713-26.
• 31. Cao G, Xing J, Xiao X, Liou AK, Gao Y, Yin XM, Clark RS, Graham SH, Chen J. Critical role of calpain I in mitochondrial release of apoptosis-inducing factor in ischemic neuronal injury. J Neurosci. 2007; 27(35):9278-93.
• 32. Bandera E, Botteri M, Minelli C, Sutton A, Abrams KR, Latronico N. Cerebral blood flow threshold of ischemic penumbra and infarct core in acute ischemic stroke: a systematic review. Stroke. 2006:37:1334 –1339.
• 33. Tsuchiya K, Kohda Y, Yoshida M, Zhao L, Ueno T, Yamashita J, Yoshioka T, Kominami E, Yamashima T. Postictal blockade of ischemic hippocampal neuronal death in primates using selective cathepsin inhibitors. Exp Neurol. 1999; 155(2):187-94.
• 34. Yoshida M, Yamashima T, Zhao L, Tsuchiya K, Kohda Y, Tonchev AB, Matsuda M, Kominami E. Primate neurons show different vulnerability to transient ischemia and response to cathepsin inhibition. Acta Neuropathol. 2002; 104(3):267-72.
• 35. Jovin TG, Yonas H, Gebel JM, Kanal E, Chang YF, Grahovac SZ, Goldstein S, Wechsler LR. The cortical ischemic core and not the consistently present penumbra is a determinant of clinical outcome in acute middle cerebral artery occlusion. Stroke. 2003;34(10):2426-33.
• 36. Dormandy TL. An approach to free radicals in medicine and biology. Acta Physiol Scand 1980,; 492:153-68. 37. Huang D, Ou B, Prior RL. The chemistry behind antioxidant capacity assays. J Agric Food Chem 2005; 53:1841–56.