- 學位論文
細胞脂噬作用對神經磷脂質失衡導致退化的保護機制
Activation of lipophagy protects neurons from neurodegeneration caused by sphingolipid imbalance
若您是本文的作者，可授權文章由華藝線上圖書館中協助推廣。
摘要
神經磷脂質 (Sphingolipid) 為神經細胞膜之重要分子，因此其胞內組成須被嚴謹調控。在果蠅神經磷脂質新合成路徑中，基因infertile crescent (ifc) 為演化上具高度保守性的脂質修飾酵素Dihydroceramide (DHC) desaturase，負責將dihydroceramide (DHC) 轉變為神經醯胺 (ceramide)。具生物活性的神經醯胺 (ceramide) 的失衡與許多神經退化疾病相關，然而關於其上游的dihydroceramid (DHC) 對於神經功能的影響仍未知。為探討ifc於神經所扮演的角色，我們製造出ifc-KO基因剔除果蠅。Sphingolipidomic analysis顯示失去ifc導致DHC增加。ifc-KO果蠅眼睛在連續光刺激下會導致感光細胞退化。透過ifc-KO感光細胞突變群組分析 (clonal analysis)，我們在連續光刺激下，觀察到脂噬 (lipophagy) 結構堆積以及活性氧物種 (ROS) 染劑H2DCF的訊號上升，因此推論DHC堆積會活化脂噬作用並誘導ROS 增加。然而，神經退化是否歸因於脂噬作用而造成細胞死亡或是活性氧物種 (ROS) 所致，則仍有待實驗證明。當減少ifc表現時，酸性胞器中的Atg8/LC3 puncta會增加，而且溶酶體的蛋白酶Cathepsins會提升，這表示活化的脂噬作用可以促進溶酶體的活性。ifc依賴性神經退化可藉由餵食抗氧化劑AD4部分拯救，表示活性氧物種 (ROS) 是造成神經退化的部分原因。此外，在ifc-KO中，活性氧物種的提升及油滴的堆積均能藉由餵食誘導細胞自噬作用發生的Rapamycin所部分抑制，因此推論在ifc依賴性神經退化細胞中提升脂噬作用，具有部分保護功能。反之，脂噬作用可藉由餵食AD4而下調，表示活性氧物種 (ROS) 為導致保護性的脂噬作用的成因。綜合上述，失去ifc會導致DHC堆積與活性氧物種 (ROS) 產生，後者隨後激活脂噬作用以保護神經免於退化。這些實驗結果支持我們的假說：DHC具有生物活性；其堆積會造成神經退化，且在這過程中細胞會活化脂噬作用以作為因應的保護機制。
並列摘要
Sphingolipids are essential membrane components of the neuron; hence their levels need to be tightly regulated. Infertile crescent (Ifc) is an evolutionarily conserved dihydroceramide (DHC) desaturase which converts DHC to Ceramide (Cer) for the de novo synthesis of Cer in Drosophila. While the imbalance of Cer, a bioactive sphingolipid, has been associated with several neurodegenerative diseases, the neuronal function of its precursor DHC remains unknown. To investigate the role of ifc, we generated ifc knockout flies (ifc-KO). Sphingolipidomic analysis showed that loss of ifc resulted in increased DHC. Prolonged light stimuli to the ifc-KO eye led to activity-dependent degeneration of photoreceptors. Clonal analysis of ifc-KO photoreceptors revealed the accumulation of lipophagic structure and the increased H2DCF signals upon light stimuli, suggesting that DHC accumulation may activate lipophagy and induce the production of reactive oxygen species (ROS). However, it remains to be determined whether the degeneration is attributed to lipophagic cell death or the ROS insults. Reduction of ifc led to the increase of Atg8/LC3 puncta in the acidified compartment and elevation of lysosomal proteases, indicating the activated lipophagy can promote subsequent lysosomal function. ifc-dependent neurodegeneration can be partially rescued by an antioxidant AD4, indicating that ROS is at least partially responsible for the degeneration. In addition, both ROS elevation and lipid accumulation in ifc-KO was suppressed by treating with the autophagy inducer Rapamycin, suggesting that enhanced lipophagy plays a protective role in ifc-dependent neurodegeneration. Conversely, lipophagy can be downregulated by AD4, indicating ROS insults lead to the feedback upregulation of protective lipophagy. In summary, loss of ifc results in DHC accumulation and ROS generation, the latter of which subsequently activates lipophagy to protect against neurodegeneration. These findings support our hypothesis that DHC is bio-active and lipophagy can be protective, highlighting their potential as therapeutic targets for regulating sphingolipid homeostasis.
參考文獻
2. Adachi-Yamada, T., T. Gotoh, I. Sugimura, M. Tateno, Y. Nishida, T. Onuki and H. Date (1999). "De novo synthesis of sphingolipids is required for cell survival by down-regulating c-Jun N-terminal kinase in Drosophila imaginal discs." Mol Cell Biol 19(10): 7276-7286.
3. Alexaki, A., S. D. Gupta, S. Majumder, M. Kono, G. Tuymetova, J. M. Harmon, T. M. Dunn and R. L. Proia (2014). "Autophagy regulates sphingolipid levels in the liver." J Lipid Res 55(12): 2521-2531.
4. Barbarroja, N., S. Rodriguez-Cuenca, H. Nygren, A. Camargo, A. Pirraco, J. Relat, I. Cuadrado, V. Pellegrinelli, G. Medina-Gomez, C. Lopez-Pedrera, F. J. Tinahones, J. D. Symons, S. A. Summers, M. Oresic and A. Vidal-Puig (2015). "Increased dihydroceramide/ceramide ratio mediated by defective expression of degs1 impairs adipocyte differentiation and function." Diabetes 64(4): 1180-1192.
5. Bartke, N. and Y. A. Hannun (2009). "Bioactive sphingolipids: metabolism and function." Journal of Lipid Research 50(Supplement): S91-S96.
6. Basu, J. and Z. Li (1998). "The Des-1 protein, required for central spindle assembly and cytokinesis, is associated with mitochondria along the meiotic spindle apparatus and with the contractile ring during male meiosis in Drosophila melanogaster." MGG - Molecular and General Genetics.