NRIP對於神經肌肉接合處的角色以及NRIP在老化中穩定神經肌肉接合處的結構

Abstract

核受體交互作用蛋白(Nuclear receptor interaction protein, NRIP)是一種依賴鈣離子的攜鈣素(Calmodulin)結合蛋白，可調節骨骼肌的收縮及再生。在先前的研究中，我們發現全身性NRIP基因剔除的老鼠表現出運動功能的缺失和遲緩的肌肉再生。此外，在16周大的肌肉NRIP基因剔除的老鼠中，呈現出受損的神經肌肉接合處(Neuromuscular junction, NMJ)，進而導致運動神經元的缺失。這些實驗結果表示NRIP可能在神經肌肉接合處的結構中扮演重要的角色。在神經突觸後的神經肌肉接合處是一種由乙醯膽鹼受體(Acetylcholine receptor, AChR)、受體相連突觸蛋白(Rapsyn)和輔機動蛋白異構體(α-actinin 2, ACTN2)所組成的複合體蛋白。而在神經肌肉接合處複合體當中，乙醯膽鹼受體是由α、β、γ和δ子單位而形成的α2βγδ五聚體，並負責接收來自運動神經元的神經傳導物質，進而使得肌肉產生收縮。在我們先前實驗的結果中，發現NRIP在骨骼肌裡與神經肌肉接合處複合體是共定位在一起，並且與AChR-rapsyn-ACTN2的神經肌肉接合處複合體有相關聯性。此外，我們先前更進一步地發現在共轉染的HEK-293T細胞中，NRIP與乙醯膽鹼受體 α子單位(AChRα)有交互作用的關係。因此，在本篇研究中，為了確定NRIP是否與乙醯膽鹼受體有直接結合的關係，我們藉由體外的pull-down assay來進一步地研究NRIP和AChRα蛋白是否有交互作用。實驗結果顯示NRIP與AChRα 蛋白在體外皆有相互作用的關係。整體而言，NRIP是一種新型的乙醯膽鹼受體結合蛋白，並且作為神經肌肉接合處複合體的架構蛋白以穩定神經肌肉接合處的結構。 另一方面，在隨著年紀增長的老鼠中，發現肌肉裡的NRIP表現量有減少的現象。此外，我們先前也發現在年老的老鼠中有運動功能缺失、異常的神經肌肉接合處以及運動神經元的損失。由於這些表現型與我們先前研究所發現的肌肉NRIP基因剔除的老鼠有相似的現象，因此，我們假設在老化的過程中，異常的神經肌肉接合處是來自於NRIP量的減少。為了驗證假說，我們研究NRIP基因治療藉由肌肉注射的方式，是否能拯救年老老鼠的神經肌肉接合處。由於先前的論文中所使用的帶有NRIP基因的AAV-DJ/8血清型的病毒在年老的老鼠中沒有表現出理想的結果，我們認為這可能是在肌肉裡NRIP表現量不足而導致的結果。因此在本研究中，我們將使用另一種適合感染肌肉的AAV9血清型病毒作為媒介，以強化先前所使用的老年老鼠中NRIP的表現量。實驗結果顯示，AAV9-NRIP 可以拯救老年老鼠的握力和神經肌肉接合處的面積，但是無法改善神經肌肉接合處的去神經化以及運動神經元的存活。整體而言，NRIP基因治療可以改善老化過程中的神經肌肉接合處的大小以及肌肉的強度。 總而言之，NRIP作為神經肌肉接合處的結構成分而成為當中新型的乙醯膽鹼受體結合蛋白，並且穩定在老化中神經肌肉接合處的結構。

Nuclear receptor interaction protein (NRIP), which is a Ca2+-dependent calmodulin binding protein, regulates the skeletal muscle contraction and regeneration. In previous study, we found that NRIP global knockout mice showed motor function deficits and delayed muscular regeneration. In addition, muscle-specific NRIP conditional knockout mice at 16 weeks of age displays impaired neuromuscular junction (NMJ), and it further leads to loss of motor neuron. These results imply that NRIP may play an important role in NMJ structure. The postsynaptic NMJ is a complex protein which is composed of acetylcholine receptor (AChR), rapsyn, and α-actinin 2 (ACTN2). In the NMJ complex, AChR consists of α, β, γ, and δ subunits to form a α2βγδ pentamer, which is responsible for receiving neurotransmitter from motor neuron to further lead to muscular contraction. In our previous results, NRIP colocalized with the NMJ complex and was associated with “AChR-rapsyn-ACTN2” NMJ complex in skeletal muscle. Moreover, it was further found that NRIP interacts with AChR α subunit (AChRα) in co-transfection of HEK 293T cells. Therefore, in this study, to determine whether NRIP directly binds to AChR, I further investigated the interaction between NRIP and AChRα proteins by an in vitro pull-down assay. The results showed that NRIP reciprocally interacted with AChRα protein in vitro. Collectively, NRIP is a novel AChR binding protein and acts as a scaffold protein in the NMJ complex to stabilize the NMJ structure. On the other hand, the decreased NRIP level in muscle was observed when age of mice advanced. In addition, there were impaired motor function, abnormal NMJ and loss of motor neuron that we previously observed in aged mice. Because these phenotypes were similar with muscle-restricted NRIP conditional knockout mice that we found in previous study. Thus, we hypothesized that abnormal NMJ is because of decreased NRIP during aging. To verify the hypothesis, we investigated whether NRIP gene therapy by intramuscular injection could rescue NMJ in aged mice. Owing to the unfavorable results in aged mice by using AAV-DJ/8 serotype virus with NRIP gene in previous thesis, we thought it might be not enough NRIP level in muscle to lead to the results. Therefore, in this study, we would use the AAV9 serotype virus, which was another suitable serotype for muscular infection, as a vehicle to boost NRIP level in previous aged mice. The results showed that AAV9-NRIP could rescue grip force and NMJ area, but it cannot improve NMJ denervation and motor neuron survival in aged mice. Collectively, NRIP gene therapy can improve the NMJ size and muscular strength during aging. In conclusion, NRIP acts as a structural component to be a novel AChR binding protein in NMJ and stabilizes NMJ structure during aging.