2014-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/657702摘要：Rab7 GTPase於真核細胞之 endolysosomal trafficking為一重要調控者。人類 Rab7點突變會導致顯性遺傳之週邊神經退化症 Charcot-Marie-Tooth 2B(CMT2B)。前人研究指出其病因可能由神經細胞中 Rab7過度活化所致。為釐清其致病機轉，我們建立了第一套 CMT2B模式動物: 首先建立 rab7基因剔除果蠅，並於其中表現造成 CMT2B之 Rab7點突變蛋白。rab7基因剔除果蠅之感覺與運動神經元均退化並喪失突觸功能。有趣的是，過度表現 Rab7突變蛋白卻可完全挽救 rab7基因剔除所導致之神經退化。本結果及相關實驗證實 CMT2B乃因 Rab7突變蛋白喪失部分功能所致。因此對 rab7表現量之調控，可為臨床治療 CMT2B患者提供新思維: 或許不須降低 Rab7活性，反而須促進其運轉機制。 為更瞭解 rab7造成神經退化之機制，我們以 rab7基因剔除果蠅為基礎進行大規模遺傳篩選。發現一基因 infertile crescent (ifc) 之過表達會促進 rab7突變體的神經退化，因此推測失去 ifc可能會抑制 rab7造成之神經退化。ifc乃演化上高度保留之酵素，負責將 dihydroceramide (DHC) 轉變成為神經醯胺 ceramide。前人於腫瘤細胞株之研究顯示，ifc表現下降會影響胞內 sphingolipids的表現量及細胞凋亡途徑。我們在果蠅視神經也同樣發現 ifc突變後造成細胞數減少及類似凋亡現象。ifc突變果蠅具有 DHC表現量上升、endosome堆積及突觸傳導缺失。有趣的是，失去 ifc反而促進溶酶體之分解能力，因此推論: ifc 之表現量在 rab7 所導致神經退化疾病中，可能扮演保護神經之關鍵角色。 為深入探討 ifc對於神經退化調控之分子機制，擬定以下目標: 一: 以遺傳學、細胞學及電生理方法，釐清 ifc與 rab7之交互作用。 二: 釐清 ifc所造成細胞死亡及神經退化之詳細機轉。 三: 探討細胞內神經醯胺等脂質含量不平衡造成細胞毒性之確切機制。 四: 找出其他調控 rab7和 ifc依賴型神經退化的基因。 本計畫結合遺傳學、生化學、顯微鏡、及電生理方法，期能使吾人釐清 rab7和 ifc所調控之神經退化機制。藉由更清楚地瞭解週邊神經退化症 CMT2B成因及分子機轉，可作為發展臨床治療方式之理論基礎。<br> Abstract: The small GTPase Rab7 is a master regulator of endolysosomal trafficking in all eukaryotic cells. Various mutations of rab7 are linked with the dominant neuropathy Charcot-Marie-Tooth 2B (CMT2B) by a previously thought neuron-specific gain-of-function mechanism. Recently we have established the first animal model for CMT2B by generating the rab7 null allele and a set of inducible transgenes expressing mutants relevant to CMT2B in Drosophila. We show that loss of rab7, but not expression of the CMT2B mutants, causes adult-onset loss of synaptic function and subsequent neurodegeneration. In sensory and motor neurons in vivo, all CMT2B proteins retain partial wild type function based on quantitative imaging, electrophysiological and viability analyses. Consequently, overexpression of the CMT2B mutants fully rescues the neuropathy-like pathologies of the rab7 null mutant with no toxic effects. The fly model indicates that disease-relevant CMT2B phenotypes may arise from partial loss of rab7 function, hence further suggests a potential therapeutic approach by stimulating rather than reducing Rab7 pathway in CMT2B patients. To discover the mechanism underlying rab7-dependent neuropathy, we perform a modifier screen for rab7-interacting genes in Drosophila. infertile crescent (ifc) is identified as a potential suppressor whose over-expression promotes the degenerative phenotypes caused by heterozygous rab7 mutant. Ifc is a highly conserved lipid-modifying enzyme that converts dihydroceramide (DHC) into ceramide. Previous literatures show that ifc knockdown impacts sphingolipids and apoptosis in human cancer cell lines. Consistent with this finding, here we show loss of ifc leads to cell loss and apoptosis-related pathologies in Drosophila sensory neurons. Furthermore, ifc mutant leads to an increase in the cytotoxic lipid DHC, endosomal accumulations, and synaptic transmission defects. Surprisingly, loss of ifc enhances lysosomal degradative capacity for transmembrane proteins; raising the possibility that the level of Ifc may be critical for neuro-protection in counteracting rab7-dependent degeneration. To understand the mechanism by which ifc regulates neurodegeneration, the following Specific Aims will be pursued: Aim 1: To characterize the subcellular dynamics and genetic relationship between ifc and rab7; Aim 2: To identify and manipulate the cell biological mechanism of ifc-mediated cell death and neurodegeneration; Aim 3: To investigate the mechanism by which imbalanced lipid composition causes neuro- toxicity; Aim 4: To identify additional genes mediating rab7- and ifc-dependent neurodegeneration. By combining genetics, biochemistry, confocal imagining, and electrophysiology, we seek to shed light on the molecular mechanisms of the identified genes. These genes may serve as potential therapeutic targets for treatment in patients with peripheral neuropathy.