THE EFFECTS OF DIFFERENT SONICATION METHODS ON ALPHA-SYNUCLEIN PRE-FORMED FIBRILS

Year 2024, , 100 - 117, 30.07.2024

Hilal Akyel , Elham Bahador Zırh , Selim Zırh , Banu Cahide Tel

https://doi.org/10.18036/estubtdc.1386713

Abstract

Alpha-synuclein (α-syn) aggregation is associated with neuronal death and the pathological hallmark of Parkinson's disease (PD). The α-syn preformed fibril model (α-syn-PFFs), reflects α-syn aggregation and is currently used in PD studies. To pass through the cell membrane, long fibrils should be fragmented by sonication. In our study, the effects of temperature, pulse modifications and/or device type on the sonication of α-syn-PFFs were investigated. Sonication was performed ultrasonic bath and in laminar-flow cabinet with probe sonicator. Dilutions were made from 5 µg/µl α-syn-PFFs stock in sterile-filtered dH2O to a final concentration and volume of 0.1 µg/µl and 200µl, respectively. Sonication was performed in an ultrasonic bath containing water at 10°C for 1 hour. All probe sonications were performed at 30% amplitude for 1 minute and 20 repetitions. The effect of temperature on sonication has been evaluated by performing sonication at room temperature (RT), in ice and in ice surrounded by dry ice. Also, the effects of pulse duration on sonication were evaluated using pulse durations of 1second(sec) on/1sec off, 3sec on/3sec off and 5sec on/5sec off. Furthermore, by waiting one minute between each sonication cycle, the heat released by the probe was prevented from affecting the fibrillar structure. The particle size was measured in triplicate by dynamic light scattering method. For transmission electron microscopy, formvar/carbon-coated grids were run through ddH2O-sonicated fibril-uranyl acetate solutions and kept dry until examined. Due to the variation in breakage of long α-syn fibrils, the effect of different parameters on sonication was investigated. In comparison of pulse durations, 5sec on/5sec off application produced shorter fibrils. Comparing the temperature interventions, lowering the temperature decreased the fibril size at 1sec on/1sec off settings but increased it at 3sec on/3sec off and 5sec on/5sec off. However, the shortest fibrils were obtained by sonication for 5sec on/5sec off at RT

Keywords

Sonication, Alpha-synuclein, Pre-formed fibril, Particle size, Transmission electron microscopy

FARKLI SONİKASYON METOTLARININ ALFA-SİNÜKLEİN PRE-FORMED FİBRİLLERİ ÜZERİNE ETKİLERİ

Abstract

Alfa-sinüklein (α-sin) agregasyonu, nöronal ölümle bağlantılıdır ve Parkinson hastalığının (PH) patolojik belirtecidir. PH araştırmalarında α-sin agregasyonunu yansıtan α-sin pre-formed-fibril (α-sin-PFF) modeli güncel olarak kullanılmaktadır. Hücre zarından geçmek için uzun fibrillerin sonikasyonla kırılması gerekir. Çalışmamızda sıcaklığın, pulse değişkenlerinin ve/veya cihaz tipinin α-sin-PFF'lerin sonikasyonu üzerindeki etkileri araştırıldı. Sonikasyon, ultrasonik banyoda ve laminar akış kabininde prob sonikatörde gerçekleştirildi. 5 µg/µl α-sin-PFF stoğundan steril-filtrelenmiş dH₂O ile sırasıyla son konsantrasyon ve hacim 0.1 µg/µl ve 200 µl olacak şekilde seyreltme yapıldı. Sonikasyon, 10°C'de su içeren ultrasonik banyoda 1 saat gerçekleştirildi. Tüm prob sonikasyonları %30 amplitüdde, 1 dakika boyunca ve 20 tekrarlı gerçekleştirildi. Sıcaklığın sonikasyon üzerindeki etkisi, sonikasyonun oda sıcaklığında (RT), buzda ve kuru buzla çevrili buzda gerçekleştirilmesiyle değerlendirildi. Ayrıca, pulse süresinin sonikasyon üzerindeki etkileri, 1 saniye(sn) açık/1 sn kapalı, 3 sn açık/3 sn kapalı ve 5 sn açık/5 sn kapalı pulse süreleri kullanılarak değerlendirildi. Ayrıca her sonikasyon tekrarı arasında bir dakika beklenerek probun açığa çıkardığı ısının fibriler yapıyı etkilemesi engellendi. Partikül büyüklüğü, dinamik ışık saçılımı yöntemiyle üç tekrarlı ölçüldü. Transmisyon elektron mikroskobu için formvar/karbon kaplı gridler, ddH₂O ile sonike fibril-uranil asetat çözeltilerinden geçirildi ve incelenene kadar kuru tutuldu. Uzun α-sin fibrillerinin kırılmasındaki varyasyonlar nedeniyle farklı parametrelerin sonikasyon üzerindeki etkisi araştırıldı. Pulse süreleri karşılaştırıldığında, 5 sn açık/5 sn kapalı uygulamasında daha kısa fibriller elde edildi. Farklı ortam sıcaklıkları karşılaştırıldığında ise ortam sıcaklığının azalması 1 sn açık/1 sn kapalı uygulamasında fibril boyutunu azaltırken, 3 sn açık/3 sn kapalı ve 5 sn açık/5 sn kapalı uygulamasında artırmıştır. Fakat en kısa fibriller 5 sn açık/5 sn kapalı ve oda sıcaklığında yapılan sonikasyon ile elde edilmiştir.

Keywords

sonikasyon, alfa-sinüklein, pre-formed fibril, partikül büyüklüğü, transmisyon elektron mikroskobu

References

• [1] Poewe W, Seppi K, Tanner CM, Halliday GM, Brundin P, Volkmann J, Schrag AE, Lang, AE. Parkinson disease. Nat Rev Dis Primers 2017; 3: 1–21.
• [2] Kalia LV, Lang AE. Parkinson’s disease. The Lancet 2015; 386(9996): 896–912.
• [3] Spillantini MG, Schmidt ML, Lee VMY, Trojanowski JQ, Jakes R, Goedert M. a-Synuclein in Lewy bodies. Nature 1997; 388: 839–840.
• [4] Lashuel HA, Overk CR, Oueslati A, Masliah E. The many faces of α-synuclein: From structure and toxicity to therapeutic target. Nat Rev Neurosci 2013; 14(1): 38–48.
• [5] Sulzer D, Edwards. RH. The Physiological Role of α-Synuclein and Its Relationship to Parkinson’s Disease. J Neurochem 2019; 150(5): 475–486.
• [6] Bartels T, Choi JG, Selkoe DJ. α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Nature 2011; 477(7362): 107–111.
• [7] Burré J, Sharma M, Südhof TC. α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation. PNAS 2014;, 111(40): 4274–4283.
• [8] Rocha EM, De Miranda B, Sanders LH. Alpha-synuclein: Pathology, mitochondrial dysfunction and neuroinflammation in Parkinson’s disease. Neurobiology of Disease 2018; 109: 249–257.
• [9] Goedert M, Jakes R, Spillantini MG. The Synucleinopathies: Twenty Years on. J Parkinsons Dis 2017; 7: 53–71.
• [10] Miraglia F, Ricci A, Rota L, Colla E. Subcellular localization of alpha-synuclein aggregates and their interaction with membranes. Neural Regen Res 2018; 13(7): 1136–1144.
• [11] Tuttle MD, Comellas G, Nieuwkoop AJ, Covell DJ, Berthold DA, Kloepper KD, Courtney JM, Kim JK, Barclay AM, Kendall A, et al. Solid-state NMR structure of a pathogenic fibril of full-length human α-synuclein. Nat Struc Mol Biol 2016; 23(5): 405–417.
• [12] Brás IC, Outeiro TF. Alpha-synuclein: Mechanisms of release and pathology progression in synucleinopathies. Cells 2021; 10(2): 1–19.
• [13] Mao X, Ou MT, Karuppagounder SS, Kam TI, Yin X, Xiong Y, Ge P, Umanah GE, Brahmachari S, Shin JH, et al. Pathological α-synuclein transmission initiated by binding lymphocyte-activation gene 3. Science 2016; 353(6307).
• [14] Masaracchia C, Hnida M, Gerhardt E, Lopes da Fonseca T, Villar-Pique A, Branco T, Stahlberg MA, Dean C, Fernández CO, Milosevic I, et al. Membrane binding, internalization, and sorting of alpha-synuclein in the cell. Acta Neuropathol Commun 2018; 6(1): 79.
• [15] Luk KC, Song C, O’Brien P, Stieber A, Branch JR, Brunden KR, Trojanowski JQ, Lee VMY. Exogenous α-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells. PNAS 2009; 106(47): 20051–20056.
• [16] Luk KC, Kehm VM, Zhang B, O’Brien P, Trojanowski JQ, Lee VMY. Intracerebral inoculation of pathological α-synuclein initiates a rapidly progressive neurodegenerative α-synucleinopathy in mice. J Exp Med 2012; 209(5): 975–988.
• [17] Volpicelli-Daley LA, Luk KC, Patel TP, Tanik SA, Dawn M, Stieber A, Meany DF, Trojanowski JQ, Lee VM. Exogenous α-Synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron 2011; 72(1): 57–71.
• [18] Volpicelli-Daley LA, Luk KC, Lee VMY. Addition of exogenous α-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous α-synuclein to Lewy body and Lewy neurite-like aggregates. Nat Protoc 2014; 9(9): 2135–2146.
• [19] Thakur P, Breger LS, Lundblad M, Wan OW, Mattsson B, Luk KC, Lee VMY, Trojanowski JQ, Björklund A. Modeling Parkinson’s disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain. PNAS 2017; 114(39): 8284–8293.
• [20] Gegg ME, Verona G, Schapira AHV. Glucocerebrosidase deficiency promotes release of α-synuclein fibrils from cultured neurons. Hum Mol Genet 2020; 29(10): 1716–1728.
• [21] Ueda J, Uemura N, Sawamura M, Taguchi T, Ikuno M, Kaji S, Taruno Y, Matsuzawa S, Yamakado H, Takahashi R. Perampanel Inhibits α-Synuclein Transmission in Parkinson’s Disease Models. Mov Disord 2021; 36(7): 1554–1564.
• [22] Carta AR, Boi L, Pisanu A, Palmas MF, Carboni E, De Simone A. Advances in modelling alpha-synuclein-induced Parkinson’s diseases in rodents: Virus-based models versus inoculation of exogenous preformed toxic species. J Neurosci Methods 2020; 338: 108685.
• [23] Karpowicz RJ, Trojanowski JQ, Lee VMY. Transmission of α-synuclein seeds in neurodegenerative disease: recent developments. Lab Invest 2019; 99(7): 971–981.
• [24] Patterson JR, Polinski NK, Duffy MF, Kemp CJ, Luk KC, Volpicelli-Daley LA, Kanaan NM, Sortwell CE. Generation of alpha-synuclein preformed fibrils from monomers and use in vivo. J Vis Exp 2019; 2(148): 1–10.
• [25] Polinski NK, Volpicelli-Daley LA, Sortwell CE, Luk KC, Cremades N, Gottler LM, Froula J, Duffy MF, Lee VMY, Martinez TN, et al. Best practices for generating and using alpha-synuclein pre-formed fibrils to model Parkinson’s disease in rodents. J Parkinsons Dis 2018; 8(2): 303–322.
• [26] Kumar ST, Donzelli S, Chiki A, Syed MMK, Lashuel HA. A simple, versatile and robust centrifugation-based filtration protocol for the isolation and quantification of α-synuclein monomers, oligomers and fibrils: Towards improving experimental reproducibility in α-synuclein research. J Neurochem 2020; 153(1): 103–119.
• [27] Pieri L, Chafey P, Le Gall M, Clary G, Melki R, Redeker V. Cellular response of human neuroblastoma cells to α-synuclein fibrils, the main constituent of Lewy bodies. Biochim Biophys Acta 2016; 1860(1): 8–19.
• [28] Tarutani A, Suzuki G, Shimozawa A, Nonaka T, Akiyama H, Hisanaga SI, Hasegawa M. The effect of fragmented pathogenic α-synuclein seeds on prion-like propagation. J Biol Chem 2016; 291(36): 18675–18688.
• [29] Singh V, Castellana-Cruz M, Cremades N, Volpicelli-Daley, LA. Generation and sonication of α-synuclein fibrils. Protocols.io 2020; 1–13.
• [30] Ryan T, Bamm V V, Stykel MG, Coackley CL, Humphries KM, Jamieson-Williams R, Ambasudhan R, Mosser DD, Lipton SA, Harauz G, et al. Cardiolipin exposure on the outer mitochondrial membrane modulates α-synuclein. Nature Communications 2018; 9(1): 1–17.
• [31] Creed RB, Memon AA, Komaragiri SP, Barodia SK, Goldberg MS. Analysis of hemisphere-dependent effects of unilateral intrastriatal injection of α-synuclein pre-formed fibrils on mitochondrial protein levels, dynamics, and function. Acta Neuropathol Commun 2022; 10(1): 1–19.
• [32] Afitska K, Fucikova A, Shvadchak VV, Yushchenko DA. α-Synuclein aggregation at low concentrations. Biochim Biophys Acta Proteins Proteom 2019; 1867(7–8): 701–709.
• [33] Kaplan M, Öztürk K, Öztürk SC, Tavukçuoğlu E, Esendağlı G, Calis S. Effects of particle geometry for PLGA-based nanoparticles: prepation and in vitro/in vivo evaluation. Pharmaceutics 2023; 15(1): 175.
• [34] Mahul-Mellier AL, Vercruysse F, Maco B, Ait-Bouziad N, De Roo M, Muller D, Lashuel HA. Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death. Cell Death and Differentiation 2015; 22(12): 2107–2122.
• [35] Mahul-Mellier AL, Burtscher J, Maharjan N, Weerens L, Croisier M, Kuttler F, Leleu M, Knott GW, Lashuel HA. The process of Lewy body formation, rather than simply α-synuclein fibrillization, is one of the major drivers of neurodegeneration. PNAS 2020; 117(9): 4971–4982.
• [36] Xue WF, Hellewell AL, Gosal WS, Homans SW, Hewitt EW, Radford SE. Fibril fragmentation enhances amyloid cytotoxicity. J Biol Chem 2009; 284(49): 34272–34282.
• [37] Shvadchak VV, Claessens MMAE, Subramaniam V. Fibril breaking accelerates α-synuclein fibrillization. J. Phys. Chem. B 2015; 119(5): 1912–1918.
• [38] Redmann M, Wani WY, Volpicelli-Daley L, Darley-Usmar V, Zhang J. Trehalose does not improve neuronal survival on exposure to alpha-synuclein pre-formed fibrils. Redox Biology 2017; 11: 429–437.
• [39] Abdelmotilib H, Maltbie T, Delic V, Liu Z, Hu X, Fraser KB, Moehle MS, Stoyka L, Anabtawi N, Krendelchtchikova V, Volpicelli-Daley LA, West A. Alpha-synuclein fibril-induced inclusion spread in rats and mice correlates with dopaminergic neurodegeneration. Neurobiol Dis 2017; 105: 84-98.