開發延緩肌萎縮性脊髓側索硬化症運動神經元退化的胜肽藥物

Abstract

肌萎縮性脊髓側索硬化症 (Amyotrophic lateral sclerosis, ALS) 是一種神經系統的退化性罕見疾病，由於脊髓的運動神經元退化而逐漸無法控制肌肉，患者肌肉在沒有受到神經支配的情況下便會逐漸地萎縮與無力。根據先前的研究發現，在 ALS 病患發病前，能偵測到肌肉中的 NogoA過量表現，並且與疾病的產生具有正相關。當以過量表現 NogoA 之Sol8 肌肉細胞的conditional medium (Sol8-NogoA CM) 處理於 NSC34 運動神經元，發現神經突的生長會受到抑制。在外添加extracellular Phosphoglycerate Kinase 1 (ePgk 1) 至Sol8-NogoA CM後，ePgk1會與運動神經細胞膜上的一種膜蛋白受體Enolase 2進行作用，並透過Rac1-GTP/Pak1-T423/p38-T180/MK2-T334/Limk1-S323/Cofilin-S3 訊息傳遞路徑，來促進運動神經元神經突的生長。Pgk1的分子量較大 (48 kDa) ，我們實驗室經過domain mapping成12個short peptides，我們發現有9個縮短的胜肽添加至NSC34神經細胞培養液中會抑制p-Cofilin (生長錐崩塌指標) 的表現，其中短胜肽M08以腦腔注射至斑馬魚胚胎中，可觀察到它會促進運動神經的axonal growth，顯示由全長蛋白質設計衍生的胜肽也可具有相同的功能。我們進一步改良M08的氨基酸特性而設計了21個不同的胜肽，再利用 Tg(mnx1:GFP) 斑馬魚轉殖基因品系進行screening，其中的M039胜肽能有更好的運動神經分枝率 (axon branching)，顯示修飾過後的胜肽在斑馬魚體內更能促進運動神經元生長的能力。另外，knock down 斑馬魚C9ORF72以誘發ALS神經退化病徵，發現注射M039的組別可以比M08 rescue 額外10%的神經長度並且讓泳動距離增加。進一步地，在使用經過Sol8-NogoA CM 處理NSC34細胞模擬類似ALS疾病情況，發現M039可以比M08更佳地促進neurite outgrowth的生長程度；在NSC34 SOD1-G93A細胞中，M039能夠可以更好地抑制p-Cofilin表現。綜合上述證據，我認為M039短胜肽具有潛力成為治療ALS神經退化性疾病療效的潛力新型藥物。

Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration of motor neurons, leading to progressive muscle atrophy and weakness, ultimately resulting in respiratory failure. Previous studies revealed that neurite outgrowth was inhibited if NSC34 neural cells were incubated with conditional medium from NogoA-overexpressing Sol8 muscle cells, but improved upon the addition of recombinant phosphoglycerate kinase 1 (Pgk1). These results suggest that Pgk1 could be a potential neuroprotective therapy for ALS. However, the large molecular weight (48 kDa) of Pgk1 would be disadvantageous in large-scale purification and treatments. To address these limitations, we first performed domain mapping and obtained 12 truncated forms of Pgk1. After screening, we identified nine candidates that, when added individually to conditional medium, could reduce the expression of p-Cofilin, a growth cone collapse marker, in NSC34 cells. Among them, the short peptide M08 was selected and administered by intracerebroventricular (ICV) injection into zebrafish embryos from the transgenic line Tg(mnx1:GFP) expressing GFP in motor neurons. We found that similar to full-length Pgk1, the axonal growth of motor neurons was significantly improved. Next, we further modified the amino acid composition of M08 and identified a short peptides, M039, which, when again administered by ICV injection into zebrafish embryos, resulted in more axonal branching than that caused by M08 administration. Additionally, in C9ORF72-knockdown zebrafish induced ALS-like neurodegenerative phenotypes, we found that M039 injection could rescue 10% higher of neurite length and increase swimming distance compared to those of M08-injected zebrafish. Moreover, significant improvement of neurite outgrowth was also found in NSC34 cells cultured in conditional medium supplemented with M039, suggesting that these modified peptides were more effective in promoting motor neuron growth when compared to the efficacy of original M08. In NSC34 SOD1-G93A cells, administration of M039 led to more efficiently suppress on the expression of p-Cofilin. Based on the above evidence, We believe that M039 has the potential to be a new peptide drug for the treatment of ALS neurodegenerative diseases.