- 學位論文
嚴重型脊髓肌肉萎縮症小鼠中運動神經元在神經肌肉交接處神經支配表徵
Characterization of Motoneuron Innervation of Neuromuscular Junction on a Severe Spinal Muscular Atrophy Mouse Model
摘要
研究背景: 脊髓肌肉萎縮症(Spinal muscular atrophy, SMA) 是一種缺乏運動神經元 (survival motor neuron protein, SMN ) 蛋白的體隱性遺傳神經肌肉疾病。SMN蛋白的缺乏可能導致突觸的缺陷及去神經化現象，最近的研究顯示，在SMN Δ7脊髓肌肉萎縮症老鼠模型上發現神經肌肉接合點(neuromuscular junctions, NMJs) 的嚴重缺陷可能與SMA有關。本論文嘗試探討台灣脊髓肌肉萎縮症小鼠模型(Type I; Smn-/-; SMN2)中神經肌肉接合點的特徵。 實驗方法: 為觀察台灣脊髓肌肉萎縮症小鼠模型(Type I; Smn-/-; SMN2)中神經肌肉接合點的缺陷與否，我們收集出生後5天到9天的小鼠肌肉。肌肉檢體包含軀幹及四肢的肌肉包括: 腓腸肌 (gastrocnemius)、脛骨前肌 (tibialis anterior, TA)、比目魚肌 (soleus)、趾長伸肌 (extensor digitorum longus,EDL)、後鋸下肌 (serratus posterior inferior, SPI)、夾肌 (splenius)、背最長肌 (longissimus)、屈趾短肌 (flexor digitorum brevis, FDB)。肌肉組織利用免疫螢光染色法，包括以抗神經絲抗體(anti-neurofilament antibody)及抗突觸體素抗體(anti-synaptophysin antibody) 來染神經末梢及α-銀環蛇毒素蛋白(α-bungarotoxin protein)來染乙酰膽鹼受體(acetylcholine receptors, AChRs)，並以共軛焦顯微鏡來觀察神經肌肉接合點的形態。為了證明去神經化現象可經由治療回復，我們在SMA小鼠出生第1天(P0)利用皮下注射給予特定序列之反義核醣核酸(antisense oligonucleotides, ASO)來評估其治療效果。 實驗結果:在SMA小鼠中FDB肌肉有超過五成嚴重的去神經支配 (>50%完全去神經終板)，而且隨著年齡增加(P5到P9)此現象有更嚴重的趨勢。給予ASO治療之後可減少去神經化現象，而達到治療的效果。除此之外，我們發現在SMA小鼠中突觸體素(synaptophysin)的訊號分布，較少於神經肌肉接合點，這可能代表SMA小鼠於神經介質的運輸有缺陷。 研究結論: 我們的研究發現，在台灣SMA小鼠模型的神經肌肉接合點中有去神經化現象，且突觸的維持和突觸體素的運輸可能有缺陷。經由治療可以恢復此去神經化現象，因此這個現象可以成為SMA治療藥物的篩選平台。
並列摘要
Background: Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease caused by a deficiency of the survival motor neuron (SMN) protein, subsequently may lead to synaptic defect and neurodegeneration. Recent studies have determined that defect in neuromuscular junctions (NMJs) are involved with SMA pathogenesis in SMN Δ7 SMA mouse model. However, the NMJs in Taiwanese SMA mouse model have not been fully investigated. Methods: To identify the NMJs of Taiwanese SMA mouse model (Type I; Smn-/-; SMN2), we collected the muscle samples from P5 to P9 mice. Muscles were dissected and labeled with anti-neurofilament and anti-synaptophysin antibodies for nerve terminals and conjugated α-bungarotoxin protein for acetylcholine receptors (AChRs). The investigated muscles include gastrocnemius, tibialis anterior (TA), soleus, extensor digitorum longus (EDL), serratus posterior inferior (SPI), splenius, longissimus, and flexor digitorum brevis (FDB). To investigate whether the denervation in FDB muscles can be restored by treatment or not, we treated the type I SMA mice by subcutaneous injection with 80μg/g antisense oligonucleotides (ASO) at P0 and sacrificed at P9. Results: In the end stage (P9), we found severe denervation (>50% fully denervated endplates) at NMJs of FDB muscles were found. From P5 to P9, the FDB’s NMJs were severely denervated by degrees. The denervation of FDB muscle could be rescued by morpholino antisense oligonucleotides (Mo-ASOs) treatment. In addition, in SMA mice, synaptophysin was rarely distributed at endplate compared to wild type, suggesting that vesicle transport may be affected at the end stage. Conclusions: Our findings indicate that the denervation in NMJs, defects both in synapse maintenance and synaptophysin transport in Taiwan type I SMA mouse model. Moreover, this phenomenon may provide a platform for rapid screening of potential drugs for SMA.