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

Yıl 2024, Cilt: 13 Sayı: 2, 100 - 117, 30.07.2024

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

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

Öz

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

Anahtar Kelimeler

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

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

Öz

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.

Anahtar Kelimeler

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

Kaynakça

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