探討肌肉 NRIP 對神經肌肉突觸和運動神經元發育的影響以及對橫紋肌肉瘤的作用

Abstract

核受體結合蛋白(NRIP)，也被稱為DCAF6和IQWD1，是一種多功能蛋白，通過與核受體和其他細胞成分的相互作用參與多種細胞的生理代謝過程。NRIP是由860個胺基酸所組成，具有7個WD40以及1個IQ結構，在先前的研究中發現NRIP會增強雄激素和糖皮質激素受體的轉錄活性，並且對肌肉完整性以及發育有重要的影響力。當NRIP在肌肉被專一性的敲除後，小鼠在6周大時出現肌肉異常但神經肌肉突觸 (NMJ) 以及運動神經元都維持正常。然而，在16周齡時，肌肉NRIP專一性敲除鼠 (MCK-NRIP cKO mice)的肌肉、肌肉神經突觸與運動神經元都出現異常的現象。 本研究主要探討了三個領域：NRIP對神經肌肉接頭（NMJ）和運動神經元發育的影響、NRIP與橫紋肌肉瘤（RMS）組織中肌生成素 (myogenin) 的表達及其關聯，以及使用免疫螢光染色實驗觀察分析NMJ的方法。 為了更深入的研究肌肉NRIP對NMJ和運動神經元發育的影響，研究首先對6、8、10、12、14、16周齡的MCK-NRIP cKO mice以及野生型小鼠 (WT mice) 進行實驗，檢測肌肉NRIP缺失對NMJ和運動神經元生理的逆行效應(retrograde effect)。免疫螢光 (IFA) 數據顯示，在14周齡時，肌肉特異性敲除NRIP的小鼠的NMJ面積相對野生型小鼠出現顯著下降，而在16周齡時，除了上述異常外，α-運動神經元數量亦顯著減少。上述異常與肌生成素表達減少有關，因為從6周齡起，肌肉NRIP缺失小鼠的肌生成素在NMJ的表達即出現顯著減少。另外，對人類橫紋肌組織進行NRIP和肌生成素的免疫染色數據顯示它們的表達存在顯著的正相關，證明了NRIP在骨骼肌中會調控肌生成素表達，並且NRIP 的缺失直接導致了肌生成素表現量下降，進一步造成NMJ 與運動神經元的異常，意即上述的逆行效應(retrograde effect)。 除了NRIP在正常肌肉生理中的作用外，我們還使用免疫組化 (IHC) 和統計分析研究了人類橫紋肌肉腫瘤 (rhabdomyosarcoma)組織中NRIP和肌生成素的表達。結果表明，NRIP在胚胎型橫紋肌肉腫瘤(embryonal rhabdomyosarcoma, ERMS)和多形性橫紋肌肉瘤 (pleomorphic rhabdomyosarcoma, PRMS)表現量較高，且NRIP的表現量會隨著橫紋肌肉腫瘤的嚴重程度加劇而上升。另一方面，肌生成素在多形性橫紋肌肉瘤中的表現相對較低，但肌生成素的表現量會隨著橫紋肌肉腫瘤的嚴重程度變大而上升。此外，研究還分析了橫紋肌肉瘤組織中NRIP和肌生成素表達的相關性。與正常肌肉組織中的結果不同，在橫紋肌肉瘤組織中兩者的表現量沒有顯著相關性，這表明癌變組織中可能存在不同的調控機制，突顯了NRIP在肌肉生理和病理中的複雜作用。 論文的最後部分提供了利用螢光染色搭配共軛焦顯微鏡觀察定量NMJ的方法。包含了小鼠解剖、分離腓腸肌、組織冷凍切片等等的詳細步驟，以及免疫螢光染色的操作方式，與共軛焦顯微鏡的設定參數，最後提供常用的軟體定量分析方法。 綜上所述，這篇論文闡明了NRIP在肌肉發育、功能和疾病中的關鍵作用。強調了NRIP通過調節肌生成素在維持NMJ和運動神經元正常生理中的重要性，並揭示了其對橫紋肌肉腫瘤的影響。同時，研究還提供了全面的NMJ觀察分析方法，從而促進了對神經肌肉疾病的進一步研究。

NRIP, also known as DCAF6 and IQWD1, is a multifunctional protein involved in various biological processes through its interaction with nuclear receptors and other cellular components. It enhances the transcriptional activity of androgen and glucocorticoid receptors in a ligand-dependent manner. NRIP, consisting of 860 amino acids with seven WD40 domains and a nuclear localization signal, plays a key role in protein-protein interactions within the nucleus. It boosts HPV-16 gene expression through glucocorticoid response elements and E2 binding sites, crucial for HPV-positive cancer cell growth. NRIP is also linked to cardiomyopathy in limb-girdle muscular dystrophy (LGMD) patients and is essential for cardiac and skeletal muscle functions. This research delved into three primary areas: the impact of NRIP on neuromuscular junction (NMJ) and motor neuron development, the NRIP and myogenin expression in rhabdomyosarcoma (RMS) tissues and their association, and the methodologies for visualizing and analyzing NMJs using immunofluorescence. To thoroughly investigate the impact of muscle NRIP on NMJ and motor neuron development, the study began by examining the retrograde effects of muscle NRIP deficiency on NMJ and motor neuron physiology at 2-week intervals, from 6 to 16 weeks of age. Immunofluorescence data showed that NRIP deficiency in muscle-specific knockout mice led to significant declines in NMJ area by 14 weeks of age and in α-motor neuron numbers by 16 weeks of age. This decline was linked to reduced myogenin expression, a key regulatory factor in muscle differentiation. Immunofluorescence assays revealed that myogenin expression at the NMJ was significantly reduced in muscle NRIP-deficient mice from 6 weeks of age. Data from immunohistochemistry staining of NRIP and myogenin on human tissue arrays showed a significant positive correlation in their expression. These results suggest that muscle NRIP's role in NMJ and motor neuron physiology is mediated through its regulation of myogenin. In addition to its role in normal muscle physiology, we investigated NRIP and myogenin expression in human RMS tissues using immunohistochemistry and statistical analysis. The results indicated that NRIP expression was significantly elevated in human RMS tissues, particularly in the embryonal and pleomorphic subtypes. In contrast, the pleomorphic subtype (PRMS) exhibited a significant decrease in myogenin expression. Moreover, the findings suggested that the expression levels of both NRIP and myogenin correlated with the severity of RMS, with higher levels observed in more advanced stages of the disease. Furthermore, the research investigated the correlation between NRIP and myogenin expression in RMS tissues. Unlike in normal muscle tissues, where NRIP and myogenin levels were significantly correlated, no significant correlation was found in RMS tissues. This suggested a distinct regulatory mechanism in cancerous tissues, highlighting the complexity of NRIP's role in muscle physiology and pathology. The final part of the thesis focused on the visualization and analysis of NMJs using immunofluorescence. The methodology involved targeting acetylcholine receptors with α-bungarotoxin to visualize NMJs in gastrocnemius muscle sections by confocal microscopy. This technique allowed for detailed imaging and quantification of NMJs, providing insights into the structural and functional integrity of neuromuscular connections. Overall, this thesis elucidated the critical roles of NRIP in muscle development, function, and disease. It underscored the importance of NRIP in maintaining NMJ and motor neuron health through myogenin regulation and revealed its potential as a biomarker for RMS severity. Simultaneously, the research provided comprehensive methodologies for NMJ visualization and analysis, thereby facilitating further studies in neuromuscular diseases.