以噬菌體展現技術開發單鏈抗體抑制α-突觸核蛋白於神經細胞內聚集之探討

Phage display-derived single-chain variable fragments targeting alpha-synuclein monomers and oligomers inhibit the aggregation of alpha-synuclein in neuron cells

游育婷(Yu-Ting Yu)

指導教授 ： 楊家榮

國立臺灣大學/醫學院/藥學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201903286

摘要

帕金森氏症是目前世界上最常見的運動障礙疾病，也是第二普遍的神經退化性疾病。患有此症的病患會有許多症狀像是不隨意顫抖、平衡困難以及無法隨意走動與移動。其中一個導致此疾病發生的病理機制為路易氏體(Lewy bodies)的不正常斑塊沉積於大腦的黑核區域。而這些不正常堆積物及是α-突觸核蛋白。α-突觸核是分子量約14至20千道耳頓的蛋白，當大量堆積在腦部時，會造成神經細胞的退化以及死亡。目前有許多對於α-突觸核蛋白於帕金森氏症上病理機轉的研究。在神經細胞中，α-突觸核蛋白會造成粒線體的功能異常、內質網的壓力上升、自噬細胞及溶小體功能異常以及突觸功能異常等等。而α-突觸核蛋白的二聚體及多聚體等會在腦中慢慢形成有毒性的纖維性α-突觸核，而這些聚集物對神經細胞皆會造成毒性與氧化壓力的上升。
本研究使用萊亨雞針對α-突觸核蛋白利用噬菌體展現技術開發了單鍊抗體(single-chain variable fragments)用來標的α-突觸核單體以及多聚體。從研究結果可以看到我們開發的單鍊抗體對於α-突觸核單體以及多聚體皆具有很高的親和性，且在試管實驗ThT aggregation assay中顯著抑制了α-突觸核蛋白的聚集。除此之外，在BiFC assay中，α-突觸核單鍊抗體也減少了α-突觸核在SH-SY5Y神經細胞中的聚集作用；在MPP+誘導α-突觸核蛋白聚集的細胞實驗模式中，α-突觸核單鍊抗體也達到相同的抑制效果，減少了單體、二聚體、三聚體及多聚體在SH-SY5Y神經細胞的表現量。此外，α-突觸核單鍊抗體在經緯靜脈注射到小鼠後，小鼠腦內的抗體量隨時間增加而增加，證實了我們開發的α-突觸核單鍊抗體具有穿透血腦障蔽的能力。
總體來說，我們開發的高親和性α-突觸核單鍊抗體抑制了α-突觸核蛋白於神經細胞內與細胞外的聚集作用，且也具有穿透血腦障蔽的能力，未來具有潛力應用於帕金森氏症的治療。

關鍵字

帕金森氏症 ； alpha突觸核蛋白 ； 單鍊抗體 ； 免疫療法

並列摘要

Parkinson’s disease (PD) is the most common movement disorder in the world. People suffering from the disease may have trouble such as moving or walking, tremor and difficult of balancing. One of the key pathologies is the formation of large plaques, Lewy Bodies, in substantia nigra (SN). The main component of the inclusions is alpha-syunclein (α-syn), a 14-20 kDa protein that accumulates in the neuronal cells, leading to the loss of cellular function and neuron death. It is now known that the mechanisms of α-syn toxicities is driven by mitochondria defects, endoplasmic reticulum (ER) stress, autophagic and lysosomal dysfunction, synaptic dysfunction, and so on. α-syn monomers, small dimers and oligomers are soluble pre-aggregated species that are considered to form toxic large oligomers and fibrils. Hence, these pre-aggregated species are demonstrated to participate in the pathogenesis.
In this study, we developed single-chain variable fragments (scFv) from chickens by phage-display techniques targeting α-syn monomers and oligomers. The result showed that our anti-α-syn scFvs have high binding affinity to both α-syn monomers and oligomers, and are able to inhibit the formation of aggregates in in vitro thioflavin –t (Th-t) aggregation assay. Besides, our anti-α-syn scFvs decreased the dimerization/oligomerization of α-syn in bimolecular fluorescence complementation (BiFC) assay in SH-SY5Y neuroblastoma cells. Our anti-α-syn scFvs also reduced the formation of α-syn monomers, dimers and trimers in MPP+-induced α-syn aggregation cell models. On the other hand, anti-G6 scFv antibody penetrated the blood-brain barrier after intravenous injection of the antibody to mice.
In summary, we have developed high affinity anti-α-syn scFvs targeting α-syn monomers and oligomers, and reduced the accumulations in in vitro assays and cell models, which may become a potential therapeutic application for α-syn pathologies.

並列關鍵字

Parkinson’s disease ； alpha-synuclein ； single-chain variable fragments ； immunotherapy.

參考文獻

1 Dehay, B., Bourdenx, M., Gorry, P., Przedborski, S., Vila, M., Hunot, S. et al. Targeting α-synuclein for treatment of Parkinson's disease: mechanistic and therapeutic considerations. The Lancet. Neurology (2015) 14, 855-866.

2 Lashuel, H. A., Overk, C. R., Oueslati, A. & Masliah, E. The many faces of alpha-synuclein: from structure and toxicity to therapeutic target. Nature reviews. Neuroscience (2013) 14, 38-48.

3 Global, regional, and national burden of Parkinson's disease, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. The Lancet. Neurology (2018) 17, 939-953.

4 George, S. & Brundin, P. Immunotherapy in Parkinson's Disease: Micromanaging Alpha-Synuclein Aggregation. Journal of Parkinson's disease (2015) 5, 413-424.

5 Muller, C. M., de Vos, R. A., Maurage, C. A., Thal, D. R., Tolnay, M. & Braak, H. Staging of sporadic Parkinson disease-related alpha-synuclein pathology: inter- and intra-rater reliability. Journal of neuropathology and experimental neurology (2005) 64, 623-628.