神經元中間絲蛋白於Dystonia musculorum小鼠背根神經節與脊髓運動神經元退化扮演角色之探討

The Role of Neuronal Intermediate Filament in the Dorsal Root Ganglion and Spinal Motor Neurons of Dystonia musculorum Mice

曾廣文(Kuang-Wen Tseng)

指導教授 ： 錢宗良

共同指導教授 ： 盧國賢

國立臺灣大學/醫學院/解剖學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.03155

摘要

Dystonia musculorum (dt)小鼠是神經型bullous pemphigoid antigen 1 (BPAG1n)基因缺損造成神經退化的突變種。前人之研究指出BPAG1n基因廣泛在各種神經組織中表現，其中背根神經節細胞會持續且大量的表達BPAG1n基因，而脊髓運動神經元則只有微弱的BPAG1n基因表現。而前人研究也發現BPAG1n這細胞骨架連結蛋白與神經元中間絲蛋白有交互作用。本論文中我們將利用不同的實驗方式，觀察神經元中間絲蛋白於dt小鼠背根神經節細胞與脊髓運動神經元所扮演的神經病理角色。
經由原位雜交與組織免疫染色實驗，我們在dt成鼠的背根神經節細胞無法偵測到BPAG1n與神經元中間絲其中一種蛋白α-internexin的存在。為了釐清缺乏α-internexin與背根神經元死亡之間的關係，因此進一步培養dt小鼠胚胎期的背根神經節細胞進行細胞細微觀察。於細胞免疫染色分析發育中的神經元發現，中間絲蛋白α-internexin無法正常送至神經纖維中，進而造成α-internexin大量堆積於細胞質或近端神經突出中，然而其他的中間絲蛋白仍是正常的分佈於神經突起。而在同時，與細胞凋亡相關之active caspase-3陽性免疫反應亦出現在大量堆積α-internexin的背根神經節細胞中。因此我們推測會與BPAG1n有交互作用的α-internexin，經由大量堆積蛋白進而影響細胞正常功能，可能是造成神經退化重要原因之一。我們推斷α-internexin在不正常堆積狀況下會阻礙神經軸突的正常運輸功能，進而使得背根神經元啟動一連串的細胞凋亡機轉。
另一方面在dt小鼠脊髓組織的免疫螢光染色實驗中，我們觀察到α-internexin不僅大量堆積在腫脹的神經纖維中，更另人意外地出現在脊髓運動神經元的細胞核內。為確認α-Internexin出現在運動神經細胞核內的現象，我們利用西方墨點法與顯微電子鏡技術進一步獲得相同映證。為了釐清α-internexin大量堆積在腫脹的神經纖維中或出現在細胞核內，是否會造成脊髓運動神經元死亡，我們進一步利用TUNEL染色分析細胞死亡，然而在運動神經元中並沒有觀察到TUNEL陽性反應。經由實驗結果證明，儘管α-internexin會大量堆積於腫脹的神經纖維中，也會出現在脊髓運動神經元的細胞核內，但是脊髓運動神經元仍然可存活下來，我們推斷這是因為脊髓運動神經元與背根神經節細胞對於α-internexin堆積的細胞耐受性並不相同，相對而言具有多極性的運動神經元對於神經元中間絲蛋白持續堆積的耐受性較佳。
總結本論文之研究，BPAG1n缺損的dt小鼠中，廣泛性神經元中間絲蛋白的堆積，會造成感覺神經元的死亡，而運動神經元卻能夠存活，因此我們推論廣泛性神經元中間絲蛋白的堆積，會導致選擇性的神經元退化。而中間絲蛋白堆積如何造成細胞死亡之機制，則值得我們未來進一步深入探討。

關鍵字

神經元 ； 中間絲蛋白 ； 背根神經節 ； 脊髓運動神經元

並列摘要

Dystonia musculorum (dt) is a mutant mouse with hereditary neuropathy. A defective neuronal form of bullous pemphigoid antigen 1 (BPAG1n) gene is responsible for this mutation. Previous study indicated that BPAG1n is expressed in wide variety of neuronal tissues and is highly and consistently expressed in dorsal root ganglia (DRG) neurons, but it is reduced in spinal motor neurons. BPAG1n is one of the cytolinker proteins that could interact with neuronal intermediate filament (IF) proteins, such as α-internexin, peripherin and neurofilaments. In the present study, we examined the pathological role of IF proteins in DRG neurons and spinal motor neurons of dt mice by using different approaches.
In our pilot study, we found that not only BPAG1n but also α-internexin was absent in DRG neurons of adult dt mice. To study the relationship between the absence of α-internexin and the progressive neuronal loss in the DRG of dt mice, we further cultured DRG neurons from embryonic dt mutants. Immunocytochemical assay of cultured DRG neurons from dt embryos revealed that α-internexin was aggregated in the juxtanuclear cytoplasm and in the proximal region of axons, yet other IF proteins were widely distributed in all processes. Active caspase-3 activity was observed in the neurons with massive accumulation of α-internexin of dt mice. From our observations, we suggest that the interaction between BPAG1n and α-internexin may be one of the key factors involved in neuronal degeneration, and abnormal accumulation of α-internexin may impair the axonal transport and turn on subsequently the cascade of neuronal apoptosis in DRG neurons of dt mice.
Immunofluorescence staining revealed that α-internexin is a major component in the swelling axon, and that an abnormal translocation of α-internexin in the nucleus of spinal motor neuron in dt mice. This abnormal translocation of α-internexin in the nucleus of spinal motor neuron was also confirmed by Western blotting and immunoelectron microscopy. Cell death of spinal motor neurons was further tested by TUNEL assay, and no positive cells could be identified from spinal motor neurons in dt mice. All these results indicated that the spinal motor neuron, unlike the DRG neuron, keep surviving after accumulation of α-internexin in the swelling axon and abnormal translocation of α-internexin in the cell nucleus. We suggest the capacity of multipolar spinal motor neurons to resist the neuronal IF aggregation is better than that of DRG neurons.
In summary, we demonstrated that massive neuronal IFs accumulations result in the cell death of DRG neuron, but not in spinal motor neuron, in dt mutants and thus we conclude that the accumulations of neuronal IFs led to selective neuronal degeneration. The mechanism of the neuronal death after the accumulation of neuronal IFs remains to be elucidated.

並列關鍵字

Neuron ； Intermediate Filament ； Dorsal Root Ganglion ； Spinal Motor Neurons

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