粒線體分裂對於辣椒素引致之神經退化之影響

Abstract

神經軸突功能及型態的完整對神經元的存活十分重要，在神經的退化過程中粒線體功能損害扮演了重要的角色，但文獻上對於粒線體型態改變對神經的影響缺少完整的研究。本研究中，我們希望透過辣椒素導致的神經退化模式了解粒線體型態變化在神經退化過程中的重要性。辣椒素在臨床上廣泛用以治療週邊神經病變引致之疼痛，其作用機制是透過活化神經細胞膜上的TRPV1蛋白引起神經退化。本研究首先建立動物模式。於小鼠腳底接受連續3日辣椒素皮下注射後以免疫組織染色方式發現皮膚末梢的表皮及真皮神經出現神經腫大(axonal swelling)，注射7日後，以電子顯微鏡觀察，發現有神經退化，並且真皮神經末梢的粒線體型態出現縮短的現象。接著以背根神經節(dorsal root ganglia)細胞培養方式進一步觀察粒線體。背根神經元的軸突在辣椒素處理後經由TRPV1活化同樣出現神經腫大及神經退化的病理現象。接著使用慢病毒(lentivirus)感染使神經元粒線體表現帶有粒線體目標序列(mitochondria-targeting sequence)的螢光蛋白作為標記，並以免疫細胞染色及活細胞觀察的方式追蹤粒線體的動態變化。於辣椒素處理後，神經軸突中靜止粒線體的長度顯著縮短。此一現象與軸突內鈣離子含量上升有關，當神經元以鈣離子螯合劑BAPTA-AM 及EDTA前處理後，粒線體的長度就不受到辣椒素處理的影響。我們進一步剖析粒線體的長度能否影響神經軸突。若讓神經元過量表現促使粒線體分裂(mitochondrial fission)的蛋白質dynamin-related protein 1 (Drp1WT)或是會抑制粒線體分離的突變蛋白Drp1K38A，軸突中的靜止粒線體會隨之減短或增加長度。於辣椒素處理後，粒線體的長短與神經腫大的嚴重程度成反比，粒線體長度越長，神經退化情況越和緩。粒線體的長短可能反映其功能缺損，因此使用TMRM染劑觀察神經軸突內粒線體的膜電位。與對照組相比，過度表現Drp1K38A的神經軸突其粒線體膜電位在辣椒素處理後仍能維持。這些研究結果顯示粒線體長度是辣椒素引致之陽離子負荷過量(cationic overload)產生神經退化的重要指標，本研究結果也建立了一個探討神經腫大及退化的模式，希冀能對臨床上常見之神經退化疾病的機制與治療提供新的方向。

Axonal degeneration is characteristics of neurodegenerative diseases and often precedes the onset of neurodegeneration. Mitochondrial function is essential for axonal integrity however it remains elusive whether and how mitochondria are actively involved in axonal degeneration by changing their sizes. In this study, we examined the role of mitochondrial dynamics in axonal degeneration induced by capsaicin, a widely used local analgesic to reduce pain sensation by activating transient receptor potential vanilloid receptor 1 (TRPV1). An intradermal capsaicin injection in vivo model and rodent dorsal root ganglia (DRG) culture in vitro model were established in the study. Axonal swellings, decreased mitochondrial stationary site length preceding axonal degeneration were manifest in rodent sensory axons upon capsaicin treatment. TRPV1-mediated axoplasmic Ca2+ increase was responsible for the alterations in mitochondrial fusion and fission. Preventing mitochondrial fission by overexpression of mutant dynamin-related protein 1 (Drp1) increased mitochondrial length, retained mitochondrial membrane potentials and reduced axonal loss upon capsaicin treatment. These results show that mitochondrial stationary site size significantly affects axonal integrity and suggest that inhibition of mitochondrial fission facilitates mitochondrial function and axonal survival following cationic overload.