以流式細胞儀進行高通量的神經突觸分析

Abstract

阿茲海默症是最常見的失智症，是一種β-澱粉樣蛋白和tau蛋白錯誤折疊的病症，也是一種失去神經元和突觸的神經退化性疾病。比起神經細胞死亡，阿茲海默症患者腦中神經末端的失去和認知功能退化更有相關性。在早期認知衰退裡，突觸功能的失調是一個關鍵。因此神經科學家使用突觸體的分離方法去研究突觸的失功能，試著找出它和阿茲海默症的關係。流式細胞術已經被許多研究團隊應用在研究突觸上，然而在突觸體的免疫染色步驟存在著一個主要的技術障礙。 傳統上突觸體的免疫染色有許多離心步驟，這些離心步驟對脆弱的胞器有累積性的傷害，這會導致大量樣品流失和訊號不穩定。我們藉由以脫硫生物素修飾突觸體表面發展出新方法以達到避免重覆離心的問題。有修飾上脫硫生物素的突觸體可被有塗佈鏈霉親合素的表面給捕捉。吸附在表面的突觸體可在溫和的條件下進行免疫染色，同時染色完還可使用倒立式顯微鏡觀察。以生物素競爭掉脫硫生物素可將突觸體從塗佈鏈霉親合素的表面給釋放出來以便於流式細胞儀的分析。這個捕捉和釋放的策略使得我們能以最少樣品量做高通量的突觸分析。 目前我們使用了三種不同的試劑，第一種是Sulfo-NHS-SS-Biotin，由於在釋放突觸體時須使用還原劑切斷雙硫鍵，同時抗體的雙硫鍵也被切斷而影響免疫染色效果，所以此試劑不適合用來修飾突觸體。第二種NHS-Desthiobiotin在釋放突觸體過程是以生物素競爭而釋放。此試劑除了修飾突觸體表面外還會進到突觸體內修飾了抗原決定位，在免疫染色時抗體可能無法辨認被修飾過的抗原決定位，導致免疫染色效果不佳。第三種Sulfo-NHS-LC-Desthiobiotin具有亞硫酸根不會穿透細胞膜進到突觸體內修飾抗原決定位，在免疫染色上有修飾過之突觸體優於未修飾突觸體，但捕捉到的突觸體數量仍是偏少，脫硫生物素和鏈霉親合素的親和力只有生物素和鏈霉親合素的親和力的千分之一，親和力是相對弱的，此外在免疫染色過程鏈霉親合素連同突觸體可能整個脫落，抑或是此試劑分子之短連接鏈尚未達到最佳化條件，所以能夠捕捉到的突觸體數量不多。綜合上述，利用脫硫生物素修飾突觸體表面發展出的新方法，還有需要改進的空間。

Alzheimer’s disease (AD) is the most common type of dementia, a protein misfolding disorder of beta amyloid and tau, and a neurodegenerative disease characterized by the loss of neurons and synapses. Loss of synaptic terminals in AD brains exhibits stronger correlations with decreased cognitive function than cell death. Disruption of synaptic function is a key event in early cognitive decline. Consequently, neuroscientists used enriched preparation of synaptosomes to study synapse dysfunction and try to find its relationship with the disease. Flow cytometry has been applied to the study of synapses by several research groups. However, the immunostaining procedure of synaptosomes prior to flow cytometry represents a major technical obstacle. Conventional immunostaining of synaptosomes for flow cytometry involves many sedimentation steps which lead to cumulative damage of these fragile organelles, resulting in significant sample loss and signal variability. We devised a method to circumvent this issue by modifying synaptic surfaces with desthiobiotin, which can be captured in wells with streptavidin-coated bottoms. Surfaced-attached synaptosomes can be immunostained under gentle conditions and imaged with inverted microscopes. Elution with biotin releases synaptic terminals from the bottom surface for flow cytometry analysis. This capture-and-release strategy enables high-throughput analysis of brain synapses with minimal tissue samples. So far, we tried three different surface modification reagents. First, we tried Sulfo-NHS-SS-Biotin(Sulfo-N-hydroxysuccinimide-Disulfide-Biotin). However, this led to poor immunostaning because the reducing agents used to cleave the disulfide bond and release the synaptosomes will also cleave the disulfide bonds of antibodies. As a result it was not suitable for modifying synaptosomes. The second modifying reagent we used was NHS-Desthiobiotin(N-hydroxysuccinimide-Desthiobiotin), as the releasing process could be accomplished by competitive elution with biotin. However, this reagent was also found to be unsuitable. It might have modified both the surface of synaptosomes and the epitopes inside the synaptosomes due to its membrane permeability, leading to poor immunostaining. The last one we used was Sulfo-NHS-LC-Desthiobiotin(Sulfo-N-hydroxysuccinimide-Long Chain-Desthiobiotin), which contained a sulfite group and thus was impermeable to cell membrane. This produced higher levels of immunostaining of labeled synaptosomes captured in wells compared to the unlabeled control group. However, the amount of captured synaptosomes was still not satisfying. This may be due to comparatively low binding affinity between desthiobiotin and streptavidin, which is only 1/1000 of that between biotin and streptavidin, or due to the detachment of streptavidin from the plate surface, or perhaps the short linker was sub-optimal for capturing. To sum up, this capture-and-release strategy still requires further optimization.