探討AAV-NRIP是否可以作爲肌萎縮側索硬化症的治療藥物

Abstract

核受體相互作用蛋白 (Nuclear interaction protein, NRIP) 也被稱爲DCAF6或是IQWD1含有860個氨基酸，其中包括了一個IQ motif 以及七個WD40 domain。我們先前的研究表示缺乏NRIP的全身性NRIP基因剔除小鼠會有肌肉無力、慢速肌凝蛋白 (slow myosin) 表現與肌肉收縮力下降以及在施打心臟毒素 (cardiotoxin) 後導致的肌肉損傷組織中有延遲的肌肉再生能力。除此之外，在肌肉NRIP基因剔除小鼠中在第6週時有異常的肌肉運動能，而在第16週時伴隨著神經肌肉接合處 (neuromuscular junction, NMJ) 異常、成肌素 (myogenin) 的下降與運動神經元 (motor neuron) 的退化。我們發現肌肉NRIP基因剔除小鼠的病徵與運動神經元疾病 (motor neuron diseases, MNDs) 相似，我們並推斷此小鼠可以成為MND的模型。因此,我們在MND中選擇了最常見的肌萎縮側索硬化症(Amyotrophic lateral sclerosis, ALS) 進行研究。我們則使用了ALS小鼠模型 (SOD1 G93A轉基因小鼠) 作為我們的NRIP治療對象。我們在先前研究發現在SOD1 G93A 小鼠中進行AAV-NRIP肌肉內註射後可以有效改善運動神經元與NMJ 數量、肌肉神經支配能力 (muscle innervation)、肌肉功能以及神經傳遞能力。這些治療結果顯示 AAV-NRIP基因治療有機會成爲ALS 疾病的治療物之一。因此在這次的研究中我們探討AAV-NRIP 如何在SOD1 G93A小鼠中實現上述的改善。 首先，NRIP已經被證明為一種可以促進肌肉細胞融合 (myoblast fusion) 的肌動蛋白 (actin) 結合蛋白。因此，我們探討了AAV-NRIP 治療後SOD1 G93A小鼠是否透過NRIP有促進myoblast fusion的功能而有肌纖維增大的現象，進而導致運動功能的改善。結果，我們通過免疫熒光分析 (Immunofluorescent assay, IFA) 觀察到AAV-NRIP 治療後的SOD1 G93A小鼠中的肌纖維大小有明顯地增加。這表示NRIP 可以通過其actin結合-myoblast fusion能力促進肌纖維增大。 此外，NRIP還可以在鈣例子的伴隨下與鈣調蛋白 (calmodulin，CaM) 結合以激活 CaN-NFATc1 和 CaMKII 途徑，而這些途徑的活化會進而促進slow myosin基因的表達。我們透過IFA探討AAV-NRIP治療後的SOD1 G93A小鼠中slow myosin表現量。我們發現 AAV-NRIP可以直接增加小鼠的slow myosin表現量以及肌纖維大小。因此這證明了NRIP能夠藉由與CaM的相互作用來增加slow myosin的表現量以達到持續性肌肉收縮能力。 NRIP能夠直接與AChR進行相互作用並穩定位於NMJ的乙醯膽堿受體 (AChR) 複合物。此複合物内含有NRIP，AChR，受體相連突觸蛋白 (rapsyn)以及輔肌動蛋白異構體 (ACTN2)。進而，我們使用了BTX-pulldown分析來研究AChR複合物之間的結合親和力是否可以通過AAV-NRIP的治療後被改善再而影響神經傳遞能力。我們的結果顯示在NRIP蛋白量下降的狀況下，SOD1 G93A 小鼠中的 AChR複合物之間的結合親和力也會降低，這證明了NRIP的缺失會擾亂NMJ的穩定性。不過，在AAV-NRIP治療後的SOD1 G93A小鼠中發現AChR複合物中只有NRIP以及AChR (而非ACTN2以及rapsyn) 之間的相互作用有被改善的現象。 此外，NRIP也可以調節myogenin的轉錄活性進而改善motor neuron的存活以及muscle innervation的能力，因此我們通過共聚焦成像評估了突觸核(synaptic nuclei)中myogenin的表現量。結果我們發現 AAV-NRIP 治療後 SOD1 G93A小鼠NMJ中的myogenin表現量有明顯增加的現象，這也表示了NRIP的確可以上調myogenin表現量以改善motor neuron的存活以及muscle innervation的能力。 總體而言，AAV-NRIP很有機會能夠成爲 ALS的治療藥物，因為NRIP可以藉由與actin結合-myoblast fusion的功能促進肌纖維增大，透過與鈣離子-CaM結合來活化下游CaN-NFATc1 途經來增加slow myosin表現量，以及上調NMJ 中的myogenin轉錄激活(transcriptional activation)。此外，NRIP的下降可能會導致 AChR 複合物之間的結合親和力降低並進而導致神經傳遞能力的降低。

Nuclear interaction protein (NRIP), which is also named DCAF6 or IQWD1 consists of 860 amino acids including one IQ motif and seven WD40 domains. Our previous studies showed that the NRIP deficiency in NRIP global knockout (NRIP gKO) mice would cause muscle weakness, reduced slow myosin expression, and muscle contraction with impaired muscle regeneration ability after cardiotoxin injury. On the other hand, muscle-specific NRIP knockout (NRIP cKO) mice show abnormal muscle function at the age of 6 weeks with abnormal NMJ structure, decreased myogenin levels, motor neuron degeneration, and motor defects at the age of 16 weeks. As the NRIP cKO mice disease phenotypes are found to resemble those of motor neuron diseases (MNDs), we hypothesized that it could be a model of MND. Hence, amyotrophic lateral sclerosis (ALS) is chosen among the MNDs for investigation and we used the ALS mouse model (SOD1 G93A transgenic mouse) as our NRIP-treating target. Previously, we found that the intramuscular injected AAV-NRIP in SOD1 G93A mice could improve the motor neuron, NMJ number, muscle innervation, muscle functions, and neurotransmission activity. These indicated that AAV-NRIP could be a potential therapeutic agent for ALS disease. In this study, we continuously investigated how AAV-NRIP could achieve these improvements in SOD1 G93A mice. First, NRIP is shown to be an actin-binding protein that could initiate myoblast fusion. Thus, we would like to discover if the increased motor function in AAV-NRIP treated SOD1 G93A mice was improved by the increase of myofiber size through myoblast fusion. As a result, we observed an increase in the myofiber size of soleus muscle from SOD1 G93A mice by AAV-NRIP treatment via performing the immunofluorescent analysis (IFA). This indicated that NRIP could increase myofiber size through its actin-binding-dependent myoblast fusion ability. Next, NRIP could also bind with calmodulin under the presence of calcium to activate CaN-NFATc1 and CaMKII pathways which might further induce slow myosin gene expressions. Thus, we further investigated the slow myosin expression level in the soleus of AAV-NRIP treated SOD1 G93A mice through IFA. We found that AAV-NRIP directly rescued the slow myosin expression level along with the increase of slow myosin myofiber size in SOD1 G93A mice. Thereof, NRIP could increase the expression level of slow myosin for continuous muscle contractions through the interaction with calmodulin. Moreover, NRIP could interact with AChR and stabilize the AChR complex (AChR-rapsyn-ACTN2) at the NMJ. Hence, we investigated whether the binding affinity between the NMJ complex could be rescued by AAV-NRIP via performing a BTX-pulldown assay for improved neurotransmission ability. Our results showed that the binding affinity between the AChR complex was reduced in SOD1 G93A under the reduction of NRIP which indicated that the loss of NRIP would disrupt the NMJ stability. However, only the interaction between NRIP and AChR but not ACTN2 and rapsyn was rescued by the AAV-NRIP treatment in SOD1 G93A mice. Furthermore, NRIP could regulate myogenin transcriptional activity, thus we evaluated the myogenin expression in synaptic nuclei which might contribute to the improved motor neuron survival and muscle innervation in AAV-NRIP treated SOD1 G93A mice through confocal imaging. We found that the myogenin expression was increased at the NMJ of AAV-NRIP treated SOD1 G93A mice which indicated that NRIP could upregulate myogenin expression for improved motor neuron survival and muscle innervation. In sum, AAV-NRIP could be a potential therapeutic drug for ALS as it increased the myofiber size through actin-binding myoblast fusion, and slow myosin expression levels through the activation of calcium-calmodulin dependent CaN-NFATc1 pathways, and myogenin expression at the NMJ through transcriptional activation. Besides, the reduction of NRIP levels could cause a decreased binding affinity between the AChR complex which might further affect disrupted neurotransmission ability.