NRIP的WD6/7可穩定神經肌肉接合處結構但無法 促進運動神經元存活以及NRIP在老化中的角色

NRIP’s fragment WD6/7 stabilizes NMJ structure but not motoneuron survival and the role of NRIP in aging

劉亦翔(Yi-Hsiang Liu)

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(2020年)

https://doi.org/10.6342/NTU202002512

摘要

NRIP也稱為DCAF6和IQWD4，NRIP是一具有860個氨基酸的蛋白質，包含一個IQ motif和七個WD 40 domain。實驗室以前的研究表明，NRIP在骨骼肌中高度表達。NRIP gKO (global knockout)小鼠在rotarod test中顯示出比野生型弱的運動功能，這是由於CaN活性，線粒體活性，CaMKII活性降低以及Ca2 +釋放減弱所致。 NRIP gKO小鼠表現出延遲的肌肉再生能力，表明NRIP在myotube formation中起重要作用。與gKO小鼠中的發現相似，NRIP cKO ( muscle-specific conditional knockout)小鼠在六週齡時表現出異常的肌肉功能。與肌肉功能相反，cKO小鼠在六週時顯示出健康的神經肌肉接合處(neuromuscular junction, NMJ)結構和運動神經元（motor neuron, MN）數量。在16週齡的cKO小鼠中，神經肌肉接合處面積顯著減少，並伴有運動神經元減少。先前的研究表明，NRIP與myogenin一起出現於synaptic region（SR）， cKO小鼠中SR的myogenin出現減少。myogenin基因治療可以挽救cKO小鼠的異常表型，此結果表明，NRIP通過myogenin參與神經肌肉接合處的維護。我們也發現NRIP可能通過充當支架蛋白來參與神經肌肉接合處的形成，NRIP可以與AChR相互作用以促進其在細胞中形成cluster。
NRIP全長、NRIP的C端（NRIP-C）和NRIP C端的WD domains(WD6/7）可以與AChR相互作用。但是當NRIP-C刪去WD7（NRIP-CΔWD7）則無法與AChR相互作用。 NRIP全長和NRIP-C皆可促進NRIP cKO小鼠體內神經肌肉接合處的結構，但是NRIP-CΔWD7無法挽救cKO小鼠的異常神經肌肉接合處結構。這些結果表明，C端的WD domain（WD7）透過與AChR相互作用可能對神經肌肉接合處形成至關重要。因此本篇，我們有興趣了解WD6/7是否可能是ALS等神經肌肉接合處缺陷疾病的潛在治療靶標。因此，我們想知道WD6/7是否可以改善NRIP cKO小鼠的異常神經肌肉接合處結構、肌肉異常、運動神經元的減少。此外，更分析了運動功能以測試WD6/7在小鼠中的治療效果。
首先，我們研究了WD6/7在NRIP cKO小鼠中的基因治療作用。WD6/7增加神經肌肉接合處大小，但無法增加神經支配和運動神經元生存。AAV-WD6/7治療無法增進NRIP cKO小鼠的運動功能。
接下來，我們研究了WD6/7和其他NRIP突變型對肌肉功能的影響。肌纖維橫截面積（CSA）中，NRIP全長明顯地增加了肌纖維CSA，而NRIP-C和WD6/7也增加了CSA。這些結果表明NRIP通過WD6/7參與的肌管形成。我們進一步研究了NRIP突變型治療小鼠中的slow myosin mRNA表現量。僅有NRIP全長能增加了slow myosin mRNA。
最後，為了瞭解為什麼WD6/7不能挽救去神經性萎縮和運動神經元的減少，因此測試了SR中myogenin的表達。與NRIP全長和NRIP-C不同，WD6/7在SR中的myogenin為下降的。NRIP的C端可以透過IQ motif誘導myogenin表達。 WD6/7缺乏誘導myogenin表達的能力，而不能透過myogenin來促進神經支配和運動神經元的存活。
總體而言，WD6/7與AChR相互作用並促進神經肌肉接合處的形成。然而，由於WD6/7不能增加slow myosin轉錄和SR中myogenin的表達，因此去神經性萎縮和運動神經元的減少不會受到影響。此外未受影響的突觸前結構導致WD6/7基因治療的NRIP cKO小鼠的運動功能障礙。

關鍵字

核受體交互作用蛋白 ； 神經肌肉接合處 ； 肌管形成 ； 肌細胞生成素

並列摘要

Nuclear interaction protein (NRIP), also named as DCAF6 and IQWD4. NRIP is a protein with 860 amino acids, containing one IQ motif and seven WD domains. Our previous study shows that NRIP mRNA is highly expressed in skeletal muscle, and expresses in the cytoplasm and localizes at the Z-disc of the sarcomere. NRIP global knockout (gKO) mice display a weaker motor function than wild-type. NRIP gKO mice exhibit delayed muscle regeneration ability, indicating that NRIP plays an essential role in myotube formation. Similar to the finding in gKO mice, NRIP muscle conditional knockout (cKO) mice show abnormal muscle function at six weeks of age.
In contrast to muscle function, cKO mice display normal NMJ structure and motor neuron (MN) numbers at six weeks. The NMJ area and innervation endplates significantly decrease in 16-week-old cKO mice, and accompanied by MNs degeneration. The results suggest a “dying back” neuropathy. The previous study shows that NRIP colocalizes with myogenin in the synaptic region (SR), and the myogenin positive nuclei in SR are reduced in cKO mice. Intramuscular myogenin gene transfer rescues the abnormal phenotypes of cKO mice. The results indicate that NRIP involves in NMJ maintenance partially via upregulating myogenin. Previously, we had found that NRIP might involve in NMJ formation by acting as a scaffold protein, NRIP could interact with AChR for cluster formation in cells.
Furthermore, NRIP mutant assays demonstrated that NRIP full length, C-terminal of NRIP (NRIP-C), and WD domains of NRIP C terminals (WD6/7) could interact with AChR; but NRIP C-terminal truncated WD7 domain (NRIP-CΔWD7) was not able to interact with AChR. NRIP-full length and NRIP-C facilitated NMJ formation delivered by AAV in NRIP cKO mice, but not NRIP-CΔWD7. These results show that the WD domain of C-terminal (WD7) might be critical to NMJ formation by interacting with AChR.
In this study, we are interested in investigating whether WD6/7 in NRIP C-terminal may be a potential therapeutic target for NMJ deficit disease like ALS. Thus, it is intriguing to know whether the functional domain WD6/7 can ameliorate the abnormal NMJ structure, muscle, and loss of MNs in NRIP cKO mice. Also, the motor functions were analyzed to examine the therapeutic effects of WD6/7 in vivo.
First, we investigated the gene therapy effects of WD6/7 in NRIP cKO mice. WD6/7 increased the NMJ size, but not nerve denervation and MN survival. Moreover, the motor function of AAV-WD6/7 treated NRIP cKO mice could not be rescued.
Next, the effects of WD6/7 and other NRIP mutants on muscle function were investigated. As to myofiber cross-sectional area (CSA), NRIP full length greatly increased the myofiber CSA, and NRIP-C and WD6/7 also increased the CSA. These results indicate the NRIP involve in myotube formation via WD6/7. We further investigated the slow-MHC mRNA level in NRIP mutants treated mice. Only NRIP full length increased the slow-MHC transcripts. Therefore, the full length of NRIP is required for inducing slow-MHC.
Finally, the myogenin expression in SR nuclei was investigated. NRIP full length and NRIP-C treatment increased myogenin expression, while WD6/7 could not induce the myogenin expression in SR nuclei. Furthermore, myogenin has been demonstrated as a neurotrophy candidate. These data suggest that WD6/7 only lacks the ability to induce myogenin expression, and cannot promote nerve innervation and MN survival through retrograde effect by inducing myogenin.
Collectively, WD6/7 interacts with AChR and promotes NMJ formation. Nevertheless, the denervated nerve terminal and loss of MN are unaffected, because WD6/7 cannot increase slow-MHC transcripts and myogenin expression in SR nuclei. Moreover, the unaffected presynaptic structure leads to motor deficits in NRIP cKO mice treated with WD6/7 gene therapy.

並列關鍵字

NRIP ； neuromuscular junction ； myotube formation ； myogenin ； WD domain

參考文獻

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