2016-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/649388摘要：infertile crescent (ifc) 是我們最近發現能調控rab7依賴性神經退化的基因。Rab7 GTPase為真核細胞之內溶酶體運輸和細胞自噬作用的重要調控蛋白，人類Rab7基因之點突變會造成顯性遺傳的週邊神經退化症Charcot-Marie-Tooth 2B (CMT2B)。為釐清其致病機轉，我們建立了第一套CMT2B之果蠅動物模式，並指出其分子機制: Rab7-CMT2B突變蛋白因無法有效與內體結合而失去部分功能，是神經突觸功能缺失因而神經退化之主因。依此論點，提高Rab7活性可治療CMT2B。為瞭解rab7突變引起退化之機制，我們以rab7基因剔除之異型合子進行遺傳篩選，發現ifc之過表達會促進神經退化，因此推測失去ifc可能會抑制退化。 Ifc為演化上高度保留的脂質修飾酵素，負責將dihydroceramide (DHC)轉變成神經醯胺。前人之癌細胞株研究發現抑制ifc哺乳類同源基因DES1，會改變磷脂質組成及影響細胞凋亡、內質網壓力和細胞自噬。然而，ifc/DES1在神經退化中之角色未明且ifc與rab7功能上的交互作用也待釐清。為探討ifc於神經中之角色，我們建立ifc基因剔除果蠅。訊息傳遞的實驗顯示失去ifc會導致發育時期神經死亡，但應非經由細胞凋亡或內質網壓力途徑。有趣的是，活體中改變ifc表現量會影響細胞自噬蛋白ATG8/LC3分布，顯示Ifc可能參與細胞自噬造成細胞死亡之途徑。因Rab7調控細胞自噬作用的多個步驟，故我們製作Ifc抗體以探討Ifc, Rab7和autophagy的胞內交互作用。功能方面，為確認神經退化過程中ifc和rab7的相互作用，將會用基因上位性分析和電生理方法來測試ifc和rab7的無功能等位基因。再者，為釐清ifc在CMT2B所扮演的角色，故我們建立新的動物模式: 內生性的CMT2B rab7K157N，其異型合子rab7K157N果蠅與CMT2B病人基因型相同，且神經呈現漸進式退化，因此可以電生理及共軛焦顯微鏡來測試Ifc對個體之影響。細胞生理方面，Ifc突變體會增加具細胞毒性之脂質DHC及內體標定物Hrs的共同堆積，顯示類神經醯胺與囊泡運輸之關聯性。有趣的是，失去 ifc會提高溶酶體分解物質能力，因此引申出一可能性: 操弄Ifc 表現量可抵抗 rab7依賴性神經退化。藉由遺傳學、生化方法、共軛焦顯微鏡及電生理，我們希望瞭解ifc於神經發育及退化之角色，以作為周邊神經退化患者可能療法的理論基礎。<br> Abstract: infertile crescent (ifc) is a gene we recently identified as a modifier for rab7-mediated neurodegeneration. The small GTPase Rab7 is a master regulator of endolysosomal and autophagic 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. However, recently we established the first animal model for CMT2B, and suggested that the adult-onset synaptic defect and subsequent neurodegeneration result from partial loss of rab7 function. Live imaging reveals that Rab7-CMT2B mutant proteins are inefficiently recruited to endosomes, but do not impair endosomal maturation. The model supports that stimulating Rab7 activity can be beneficial in treating CMT2B; hence prompts us to identify components regulating rab7-dependent degeneration. From a genetic screen, we identify ifc as a potential suppressor which overexpression promotes the degenerative defects caused by heterozygous rab7 mutant. Ifc is an evolutionarily conserved lipid-modifying enzyme that converts dihydroceramide (DHC) into ceramide. Knockdown of DES1, the mammalian homolog of ifc, has been shown to impact the composition of sphingolipids and affect apoptosis, autophagy, and ER stress in cancer cell lines. However, the cell biological role of ifc/DES1 in neurodegeneration is unknown, and the functional relationship between ifc and rab7 remains to be answered. To investigate the neuronal role of ifc, we have generated the ifc knockout fly. Our preliminary results indicate loss of ifc leads to developmental neuronal loss that is unlikely caused by apoptosis or ER stress. Interestingly, both expression and knockdown of ifc results in altered subcellular patterns of ATG8/LC3-mcherry, suggesting the regulation by Ifc in autophagic cell death. Because Rab7 regulates multiple steps in the endolysosomal and autophagic pathways, we have generated anti-Ifc antibody to reveal the subcellular interaction between Ifc, Rab7, and autophagy. To determine the genetic relationship between ifc and rab7 in neurodegeneration, we will assay the null alleles of both ifc and rab7 using genetic epistasis and electrophysiology. Furthermore, to examine the role ifc plays in CMT2B, we have engineered the endogenous knock-in of the CMT2B mutant flies rab7K157N. The heterozygous rab7K157N represents the genetic background in CMT patients, and exhibits progressive functional degeneration; therefore allows for assaying the interaction between Ifc and Rab7 by confocal imaging and electrophysiology. Finally, ifc mutant leads to increased levels of both the cytotoxic lipid DHC and the endosomal marker Hrs, indicating an intriguing link between ceramide lipids and vesicle trafficking. Interestingly, loss of ifc enhances lysosomal degradative capacity, raising the possibility that manipulating the level of Ifc may be neuro-protective in counteracting rab7-dependent degeneration. By combining genetics, biochemistry, confocal imagining, and electrophysiology, we seek to understand the neuronal role of ifc, which potentially will shed light on the therapeutic approaches for patients with peripheral neuropathy.