2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38590It is a critical goal for neuroscience researchers to study of the neuronal degeneration and apoptosis for human neurological disease. In this proposal, we would like to use the neurological mutant, dystonia musculorum (dt) mouse, for the study neurological disease, for example, Alzheimer’s disease and amyotrophic lateral sclerosis (ALS). It was reported that neurodegenerative diseases are morphologically characterized by progressive neuron loss, associated typical cytoskeleton changes, in clinical studies. The mechanisms lead to the neuronal intermediate filament (IF) cytoskeleton aggregation in the cytoplasm and the axon are still unclear, but it may due to toxic insults, overexpression neuronal IF or the failure of axonal transportation. In studies of dt mice, accumulation of neuronal IF within axons and cell bodies were also observed. This is a three year project and it includes following: The primary aim of this study is to illustrate the relationship between neuronal IF aggregation and neuronal degeneration of sympathetic neurons in dt mice. We found not only apoptosis of sensory neurons but also degeneration of sympathetic neurons from the innervations of hind paws in dt mice.We will study the neuronal degeneration of sympathetic neurons in the peripheral nervous system that mainly migrated from the neural crest during development in dt mice. To study the axonal degeneration patterns of sympathetic neurons from dt mutants will be examined at the ultrastructal level by electron microscopy. Expression patterns of apoptosis-related proteins in the programmed cell death of neurons will be also examined by immunocytochemistry andWestern blot.We will extensively examine by terminal deoxynucleotidyl transferase (TdT)-meditated dUTP-biotin nick-end labeling (TUNEL) assay as well. Furthermore, we will study the protection function of ubiquitin proteasome system in degenerating neurons with neuronal IF aggregations. The ubiquitin proteasome system is a central component in the cellular defense against potentially toxic proteins aggregates, including neuronal cytoskeleton. To determine whether the neuronal death is suppressed by ubiquitin, the p-EGFP-tagged ubiquitin and control pEGFP-N2 vector will be transfected in the sympathetic neurons from dt and wild-type mice in vitro. In addition, it could be done to demonstrate that overexpression of ubiquitin in neurons might disaggregate the neuronal IF inclusions after acrylamide intoxication in ubiquitin-EGFP-transfected stable PC12 and Neuro2A cell line models. The protein ubiquitin involved in the regulation of neuronal survival that would be demonstrated in cell culture models. Moreover, the correlations between the protection function of ubiquitin formation and degenerating neurons with neuronal IF aggregations will be investigated directly by immunofluorescenec microscopy, confocal microscopy, electron microscopy and Western blot. At the completion of this project, we will provide new information on underlying mechanism of neuronal degeneration and cell apoptosis in the development of the neurological mutants. Moreover, the protective functions of ubiquitin including anti-apoptotic and anti-protein aggregation mechanisms will be also examined in the neuropathological disease.在神經科學研究領域，探討神經細胞的退化與凋亡已成為今日生物醫學界的重要 挑戰。本計劃以神經性缺陷的突變小鼠Dystonia musculorum (dt) 做為生物研究材料， 探討台灣常見的神經性疾病，例如老年癡呆症與肌萎縮性脊髓側索硬化症。臨床上， 許多神經退化疾病都有的病理特徵，是會伴隨其細胞骨架排列的變化以及細胞骨架大 量堆積於神經元。但造成神經元中間絲蛋白這種細胞骨架蛋白堆積的機制目前尚未完 全釐清，可能因素包括:遭受毒物後的反應、神經元內不正常大量製造中間絲蛋白或神 經纖維運輸障礙等原因。而在過往dt 小鼠研究中也觀察到相同神經退化的病理特徵， 神經元或神經軸突內均有中間絲蛋白堆積的現象。 初期，將以dt 小鼠為生物研究材料，希望能瞭解中間絲蛋白堆積與交感神經元死 亡的關聯性。之前我們所發表dt 小鼠感覺神經退化的論文中，除了觀察到支配皮膚感 覺的背根神經節細胞有凋亡現象外，同時也發現到，支配汗腺的交感神經元亦嚴重的 退化，這是之前學者未觀察到的。我們將以電子顯微鏡技術研究交感神經退化時的超 微結構變化；以免疫細胞化學染色與蛋白質電泳等方式進行研究，觀察神經退化時 caspase 與其他細胞凋亡相關蛋白的表現；並利用TUNEL 染色分析細胞死亡。 進而，我們將全力研究中間絲蛋白堆積的退化神經元中，ubiquitin proteasome system 是否可藉由清除堆積蛋白的功能扮演保護性角色。細胞中ubiquitin proteasome system 可快速清除不正常堆積的蛋白質，避免對細胞造成傷害。我們分別 取dt 與正常小鼠的交感神經元進行初級細胞培養，再將ubiquitin 核酸序列末端接上綠 色螢光蛋白質的核酸序列，轉殖進入細胞中表現，觀察ubiquitin 是否在中間絲蛋白 堆積的退化神經元中可扮演保護性功能，增加細胞存活率。另外，我們亦篩選出持續 表現ubiquitin 末端接有綠色螢光蛋白(ubiquitin-EGFP)的PC12 與Neuro2A 細胞株， 建立在神經元遭受acrylamide 等毒物傷害後，ubiquitin proteasome system 扮演清 除細胞中堆積中間絲蛋白的保護性功能之細胞模式。並以免疫細胞螢光染色、共軛焦 顯微鏡技術、電子顯微鏡技術與蛋白質電泳等實驗方式，進一步探討ubiquitin 在神 經退化中所參與細胞保護機制。本計劃完成之後，吾人能對中間絲蛋白的堆積，導致交感神經退化與凋亡的機制 能提出新的見解；並利用持續表現ubiquitin 細胞株的實驗模式，探討神經退化時， ubiquitin proteasome system 在神經元所扮演的保護性角色。神經退化細胞骨架中間絲蛋白neuronal degenerationcytoskeletonintermediate filamentProject