肌肉NRIP對於神經肌肉接合處的維持之重要性探討

Muscle-derived NRIP is essential for neuromuscular junction maintenance

黃韻心(Yun-Hsin Huang)

指導教授 ： 陳小梨

國立臺灣大學/醫學院/微生物學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201802090

摘要

核受體交互作用蛋白(Nuclear receptor interaction protein, NRIP)是一個鈣離子依賴性的攜鈣素(calmodulin)結合蛋白。NRIP可以透過調節粒線體的活性與鈣離子的平衡來調控肌肉的收縮功能。在本實驗室先前的研究中，發現全身性NRIP基因剔除小鼠有肌肉失養和運動能力顯著受損的情形。另外，我們在肌肉NRIP基因剔除小鼠中證明了肌肉NRIP的缺失會導致運動神經元 (α-motor neuron)退化和神經肌肉接合處(neuromuscular junction, NMJ)的異常。此外，我們也發現NRIP和乙醯膽鹼受體(acetylcholine receptors, AChRs)共定位(co-localize)於神經肌肉接合處，並且NRIP作為一個營養因子，能藉由穩定神經肌肉接合處來支持運動神經元的存活。因此，NRIP在維持神經肌肉接合處中所扮演的角色將是我們有興趣去探討的。神經肌肉接合處複合物的結構組成包含了乙醯膽鹼受體、受體相連突觸蛋白(rapsyn)和輔肌動蛋白異構體(α-actinin2, ACTN2)。在本研究中，我們用組織免疫螢光染色法來探討在正常小鼠中NRIP與神經肌肉接合處組成分的位置。結果顯示NRIP與乙醯膽鹼受體、受體相連突觸蛋白和輔肌動蛋白異構體共定位於神經肌肉接合處，這意味著NRIP也是神經肌肉接合處複合物的結構組成分之一，並且可能在維持神經肌肉接合處完整性中扮演著重要的角色。
另一方面，肌肉NRIP基因剔除小鼠的病徵與運動神經元疾病(motor neuron diseases, MNDs)類似，例如神經肌肉接合處異常、運動神經元退化和運動能力的缺陷，暗示著肌肉NRIP可以逆行調節運動神經元的存活。根據逆行死亡假說(dying back hypothesis)，神經肌肉接合處的退化可能是導致運動功能逐漸惡化的原因，這說明了神經肌肉接合處可做為運動神經元疾病的治療標的。如前面所述，NRIP參與了神經肌肉接合處的形成，因此我們假設NRIP在運動神經元疾病中可以作為神經肌肉接合處的治療物。我們在肌肉NRIP基因剔除小鼠中，透過肌肉注射的方式來進行AAV-NRIP基因治療，以探討NRIP是否可以改善肌肉NRIP基因剔除小鼠的肌肉功能（包括肌肉氧化功能標誌物和肌肉力量）、運動神經元的存活（運動神經元數量）和神經肌肉接合處的功能〔包括神經肌肉接合處的面積和軸突神經支配/去神經支配的比例(axonal innervation/denervation)〕。值得注意的是，AAV-NRIP基因治療改善了肌肉NRIP基因剔除小鼠的慢肌凝蛋白(slow myosin)的表現、增加了神經肌肉接合處的面積與神經支配的比例，以及增強了運動神經元的存活。總結來說，AAV-NRIP可以挽救肌肉NRIP基因剔除小鼠的肌肉和運動功能。
另外，肌萎縮性脊髓側索硬化症(amyotrophic lateral sclerosis, ALS；運動神經元疾病的一種)目前尚無能完全治癒的藥物。ALS是一個運動神經元漸進性退化的疾病，並伴隨著神經肌肉接合處的異常。由於NRIP可以維持神經肌肉接合處的完整性，所以NRIP在ALS模式小鼠(hSODG93A mice)中的作用將是值得去發掘的，並且有機會能將NRIP發展成為ALS疾病的一個治療物。我們發現與正常小鼠相比之下，hSODG93A 小鼠的脊髓、腓腸肌和脛骨前肌中的NRIP蛋白表現量有顯著的下降，這意味著NRIP可能在hSODG93A 小鼠中扮演了角色。因此，我們未來將會對hSODG93A 小鼠進行AAV-NRIP基因治療來觀察是否可以改善ALS疾病的病徵。
除此之外，在我們先前的研究中有發現到隨著年紀的增長，NRIP蛋白表現量會隨之下降，並且運動能力也有顯著的變差。在本篇研究中，我們繼續透過觀察肌肉功能、運動神經元存活與神經肌肉接合處的功能來調查NRIP對老化過程的影響。結果顯示與正常的成鼠(16週齡)相比，老年小鼠(103週齡)的肌肉氧化功能異常並有運動能力的缺陷，還有運動神經元退化、神經肌肉接合處結構異常與軸突去神經支配的情況。這代表著肌肉NRIP的表現量下降可能是導致肌肉氧化能力異常、運動神經元缺失和神經肌肉接合處退化的原因之一。在未來我們將會研究AAV-NRIP基因治療是否能夠恢復老年小鼠的肌肉、運動神經元與神經肌肉接合處的功能。

關鍵字

核受體交互作用蛋白 ； 神經肌肉接合處 ； 基因治療 ； 肌萎縮性脊髓側索硬化症 ； 老化

並列摘要

Nuclear receptor interaction protein (NRIP) is a Ca2+-dependent calmodulin-binding protein. NRIP can regulate muscle contraction through mediating mitochondrial activity and Ca2+ homeostasis. In previous study, we observed that NRIP global knockout (gKO) mice show muscle dystrophy and impaired motor performance. Moreover, muscle-specific NRIP conditional knockout (cKO) mice demonstrate that the deprivation of NRIP in muscle can cause motor neuron degeneration and neuromuscular junction (NMJ) abnormality. Besides, NRIP co-localizes with acetylcholine receptors (AChRs) at NMJ and acts as a trophic factor supporting spinal motor neuron via stabilization of NMJ. Therefore, the role of NRIP at NMJ maintenance would be interesting to investigate. The structural components of NMJ complex at muscle membrane include AChRs, rapsyn and α-actinin2 (ACTN2). Hence, in this study, we investigated the location of NRIP with NMJ components in WT mice by immunofluorescence assay. Results showed that NRIP co-localized with AChRs, rapsyn and ACTN2. This implied that NRIP is also a structural component of NMJ complex, and NRIP in muscle may play an important role in maintaining NMJ integrity.
On the other hand, muscle-specific NRIP cKO mice showed the phenotypes similar to motor neuron diseases (MNDs), such as NMJ abnormality, motor neuron degeneration and motor performance defects, implying that muscle NRIP can retrogradely regulate motor neuron survival. According to dying back hypothesis, NMJ degeneration may be the contributor to the progressive deterioration of motor function, suggesting that NMJ is a therapeutic target. As mentions, NRIP is involved in NMJ formation; therefore, we hypothesized that NRIP could be a therapeutic agent at NMJ in MNDs. We performed AAV-NRIP gene therapy in NRIP cKO mice through muscle injection to examine whether NRIP can improve muscle functions (including oxidative muscle markers and muscle strength), motor neuron survival (α-motor neurons number) and NMJ functions (including NMJ size and axonal innervation/denervation) in NRIP cKO mice. Notably, AAV-NRIP gene therapy through intramuscular injection improved slow myosin expression, increased NMJ size and innervation, and enhanced motor neuron survival in muscle-specific NRIP cKO mice. In sum, AAV-NRIP can rescue muscle and motor neuron functions in NRIP cKO mice.
Additionally, there is not yet a cure treatment in amyotrophic lateral sclerosis (ALS; is one of the MNDs). ALS show progressive neurodegeneration and abnormalities at the NMJ. Due to NRIP functions for NMJ maintenance, the role of NRIP in hSODG93A mice (ALS mouse model) would be interesting to explore and it may be able to develop NRIP as a therapeutic agent for ALS. We found that the protein expression of NRIP in spinal cord, gastrocnemius (GAS) and tibialis anterior (TA) muscles were downregulated in hSODG93A mice compared with WT mice. This implied that NRIP might play a role in hSODG93A mice. Hence, we will perform AAV-NRIP therapy in hSODG93A mice in the future to investigate whether AAV-NRIP delivering can improve disease phenotypes of hSODG93A mice.
Furthermore, we previously observed that the NRIP protein level of muscle tissues was decreased with advancing age along with the declination of motor functions. In this study, we continuously examined the effect of NRIP in aging through observing muscle functions, motor neuron survival and NMJ functions. Results showed muscle abnormality with reduction of muscle oxidative functions and motor function defects in aged mice (103-week-old) compared with adult wild type mice (16-week-old). Aged mice also displayed motor neuron degeneration, abnormal architecture of NMJ and axonal denervation. This implied that the reduction of NRIP in muscle may be the cause of muscle oxidative function abnormality, motor neuron loss and NMJ degeneration. In the future, we will examine whether AAV-NRIP can restore muscle, motor neuron and NMJ function in aged mice.

並列關鍵字

NRIP ； neuromuscular junction ； gene therapy ； amyotrophic lateral sclerosis ； aging

參考文獻

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