請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49400
標題: | 肌肉NRIP表現缺失造成年齡依賴漸進性運動神經元退化和運動功能障礙作用之探討 Muscle-derived NRIP deficiency causes age-dependent progressive motor neuron degeneration and motor deficits |
作者: | Tung-Chien Wu 吳東謙 |
指導教授: | 陳小梨 |
關鍵字: | 核受體交互作用蛋白,運動神經元,神經肌肉接合處,衛星細胞,基因治療,肌肉表現因子, NRIP,motor neuron,neuromuscular junction,satellite cell,gene therapy,muscle-derived factors, |
出版年 : | 2016 |
學位: | 碩士 |
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,能夠調節肌肉細胞內粒線體的活性和鈣離子的平衡以維持正常的肌肉收縮功能。在本實驗室先前的研究中,我們發現全身性NRIP基因剃除小鼠其粒線體的功能活性明顯下降,而跑動能力也有顯著受損的情形,另外,我們發現16週大的肌肉NRIP基因剃除小鼠與同週齡的正常小鼠相比,其alpha運動神經元(alpha motor neuron, α-MN)存活數量明顯減少,神經肌肉接合處(neuromuscular junction, NMJ)結構完整性變差,軸突神經支配(axonal innervation)面積縮小,我們也發現NRIP會表現在脊髓和神經肌肉接合處中,表示肌肉NRIP對於維持alpha運動神經元存活數量和神經肌肉接合處結構完整性扮演著重要的角色。然而,對於老化的小鼠而言, 肌肉NRIP是否仍有保護的作用是一件值得去探討的問題。
我們分別取下年幼(6週大)、成年(16週大)、衰老(80週大)各階段小鼠的腰段脊隨,利用免疫螢分析法去進行神經元核蛋白(neuronal nuclear protein, NeuN)和乙醯膽鹼轉化酶(choline acetyltransferase, ChAT)抗體雙染,以計數脊髓腹側角alpha運動神經元的量;利用全組織免疫螢光染色進行螢光標定的alpha-銀環蛇毒素(α-BTX)(為神經肌肉接合處形成的標記物)和神經絲蛋白(neurofilament, NF)(為運動神經元軸突標記物)抗體雙染,去分析比目魚肌中神經肌肉接合處的型態;利用免疫組織化學染色法偵測慢肌凝蛋白(slow myosin)的表現情形,以代表肌肉的組成和發育;而前肢肌肉強度和運動行為表現則是分別透過握力測試和滾輪運動測試加以分析。 實驗結果顯示在正常小數中,NRIP表現量、alpha運動神經元數量和跑動能力皆有隨著年紀增加而下降的趨勢,表示NRIP量的減少與alpha運動神經元退化和運動功能障礙息息相關,而另一方面,各週齡的肌肉NRIP基因剃除小鼠與正常小鼠相比,alpha運動神經元缺失和運動功能異常的現象更為嚴重。此外,我們發現肌肉NRIP基因剃除小鼠,隨著年紀增長其alpha運動神經元減少和運動功能衰退的趨勢與正常的小鼠相似,表示除了NRIP之外,還有其他未知的老化因子會造成運動功能上的缺陷。綜合上述的結果,我們得知肌肉NRIP基因表現的缺失會造成年紀依賴漸進性的alpha運動神經元退化和運動功能障礙。 NRIP和衛星細胞(satellite cell)皆會參與在神經肌肉接合處發育和肌肉再生過程之中,並且在老化個體中,衛星細胞數量上的減少被報導為造成老化相關運動功能障礙的成因之一。為了檢視NRIP是否會影響衛星細胞的活化或增生,我們將心臟毒素注射到正常和肌肉NRIP基因剃除小鼠的脛前肌中並分別於1天、3天及6天後,收取該肌肉檢體進行免疫螢光染色,觀察成對盒轉錄因子7(paired box transcription factor 7, Pax7)和成肌分化因子(myogenic differentiation factor, MyoD)的表現,這兩基因消長的表現模式為判斷衛星細胞處於活化或增生和定向風化的指標。從量化的實驗結果顯示,正常小鼠肌肉的衛星細胞在肌肉受損後的第一天即開始活化或增生,並隨後在第三天進行分化的動作;但肌肉NRIP基因剃除小鼠中的肌肉衛星細胞則會延至肌肉注射後的第三天才開始活化或增生,而在第六天才有明顯的定向分化發生,意味著NRIP基因表現的缺失會延遲肌肉再生的作用過程,總而言之,NRIP會影響骨骼肌再生過程中衛星細胞的活化和分化行動。 為了更加支持肌肉NRIP對於運動神經元的重要性,我們將帶有NRIP基因的腺病毒注射至肌肉NRIP基因剃除小鼠中,探討局部注入的NRIP是否能改善肌肉失養的情形。而實驗結果顯示,給予NRIP治療後9週大的肌肉NRIP基因剃除小鼠其神經肌肉接合處的大小顯著比施打GFP的對照組大,但對於alpha運動神經元數量和運動功能表現,兩組之間並無顯著的差異,表示NRIP可視為一個肌肉表現的神經營養因子(muscle-derived neurotrophic factor)有助於神經肌肉接合處的生長與形成。 Nuclear receptor interaction protein (NRIP) is a Ca2+-dependent calmodulin-binding protein mediating mitochondrial activity and Ca2+ homeostasis both involved in muscle contraction. In previous study, we observed that NRIP global knockout (gKO) mice have reduced mitochondrial activity and impaired running ability. Moreover, NRIP muscle-specific conditional knockout (cKO) mice have impaired alpha motor neuron (α-MN) survival, neuromuscular junction (NMJ) integrity, and axonal innervation compared to aged-matched wild-type (WT) mice at the age of 16 weeks. Also, NRIP is expressed in both the spinal cord and NMJs. It implies that NRIP in muscle may play an important role in maintaining α-MN survival and NMJ integrity. However, it is interesting to know whether the protective effect of NRIP persists in aged mice. In our study, we isolated the lumbar spinal cord from juvenile (age 6 wk), adult (age 16 wk), and senescent mice (age 80 wk) to count the α-MN number in the ventral horn by IFA co-staining of anti-neuronal nuclear protein (NeuN) and anti-choline acetyltransferase (ChAT) antibody. The morphology of soleus (SOL) NMJs was analyzed by whole-mounting IFA co-staining fluorescently tagged alpha-bungarotoxin (α-BTX) (NMJ formation indicator) and anti-neurofilament (NF) (MN axon) antibody. The muscle development was checked by soleus IHC for slow myosin expression that represented a marker for muscle character. The forelimb muscle force and the motor behavior were examined by the grip strength assay and the rotarod test, respectively. Results showed that the NRIP level, α-MN number, grip strength, and running ability declined with age in WT mice. It implies that less NRIP expression is associated with α-MN degeneration and motor deficits. On the other hand, cKO mice exhibited significantly decreased α-MNs and performed severe motor dysfunction in comparison with WT at each age-matched littermates. Moreover, the declined trends of α-MN survival and motor functions with age were similar to WT. It indicates that in addition to NRIP, the unrevealed aging factors may play roles for the defection of motor functions. These data suggest that muscle NRIP deficiency causes age-dependent progressive α-MN degeneration and motor deficits. Both NRIP and satellite cells are involved in NMJ development and muscle regeneration. Also, loss of satellite cells in aged individuals is reported to be as one of contributing factors to cause age-related motor deficits. To examine whether NRIP affected satellite cells activation/proliferation, we injected cardiotoxin into WT and cKO mice tibialis anterior (TA) muscles and harvested at 1, 3, and 6 days after muscle injury to perform IFA staining anti-paired box transcription factor 7 (Pax7) and anti-myogenic differentiation factor (MyoD). Expression patterns of these two genes are indicators for satellite cell activation/proliferation and commitment differentiation. Quantitative data revealed that satellite cells in WT muscles began to activate/proliferate at day 1 and subsequently to differentiate at day 3 after damage; but cKO muscles delayed satellite cell activation/proliferation at day 3 and commitment differentiation at day 6 after injury. Collectively, deficiency of NRIP can delay muscle regeneration. In sum, NRIP affects satellite cells activation and differentiation during muscle regeneration in skeletal muscles. Additionally, to further support the role of NRIP in muscles and motor neuron functions, we injected cKO mice NRIP encoding by adenovirus at the age of 6 weeks to determine whether NRIP could rescue muscle dystrophy. Results showed that cKO mice treated with ad-NRIP had a larger size of NMJs than relative control (ad-GFP) at age 9 wk. However, there was no significant difference in the α-MN number and motor performance between ad-NRIP and ad-GFP treatment group. It implies that NRIP is a muscle-derived neurotrophic factor for NMJ growth. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49400 |
DOI: | 10.6342/NTU201603184 |
全文授權: | 有償授權 |
顯示於系所單位: | 微生物學科所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-105-1.pdf 目前未授權公開取用 | 2.1 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。