Rac1-NOX途徑產生之活性氧物種於果蠅視神經調控 活性依賴神經退化之機制

Abstract

神經磷脂質(Sphingolipid)為神經細胞膜之重要分子。在果蠅的神經磷脂質新合成路徑中，Infertile crescent (Ifc)為演化上具高度保守性的脂質修飾酵素–Dihydroceramide desaturase，具有轉化二羥基神經醯胺(dihydroceramide, DHCer)成神經醯胺(ceramide, Cer)的能力。之前實驗發現，在果蠅視神經失去ifc，會造成漸進式活性依賴退化、活性氧物質增加，以及細胞自噬活化的現象。 為探討活性氧物質的生成機制，前人在眼睛具有ifc-KO全感光細胞突變群組(ifc-KO whole eye clone)的果蠅的食物中餵食藥物apocynin去抑制NADPH oxidase (NOX)，發現可以減緩視神經失去ifc所產生的神經退化。我利用ifc-KO感光細胞突變群組分析進而發現，Rac1在ifc-KO細胞中活化。同時，藉由視神經電位紀錄(electroretinogram)測試果蠅視神經的功能得知，在失去ifc的視神經中，再去抑制Rac1或是NOX，皆可部份恢復神經退化，因此我們認為Rac1-NOX為果蠅眼睛失去ifc之下，活性依賴退化及活性氧物種產生的途徑之一。為了進一步觀察Ifc和Rac1的關係，我藉由免疫共沉澱的方法發現兩者並沒有產生物理性交互作用，表示兩個蛋白的交互作用低於西方墨點法可檢測到的範圍，或是他們之間需要其他蛋白的溝通才能互相影響，須待進一步的實驗證明。 之前實驗發現，Ifc和Autophagosome 於視神經有共位的現象；同時，經由序列預測發現Ifc和autophagosome有四個可能產生連結的區域-LC3-interacting region (LIR)。因此我藉由免疫共沉澱的方法，進一步觀察兩者的關係，發現Ifc和autophagosome有產生物理性交互作用。另外，我們發現將同時突變四個LIRs的Ifc放回ifc-KO同型合子的果蠅時，無法拯救失去Ifc所造成的死亡。因此推論，存在於autophagosome的Ifc蛋白對果蠅的生存極為重要。 這些實驗結果支持:在果蠅視神經失去ifc，細胞會經由Rac1-NOX途徑產生活性氧物質及活性依賴退化。此外，存在於autophagosome的Ifc蛋白，對果蠅的生存有重大的影響。

Sphingolipid is an essential component of cell membrane. Infertile crescent (Ifc) is an evolutionarily conserved dihydroceramide desaturase, which converts dihydroceramide to ceramide. Previously we have found that loss of ifc in Drosophila photoreceptors results in activity-dependent neurodegeneration, elevation of reactive oxygen species (ROS), and activation of autophagy. To investigate the source of ROS, a NADPH oxidase (NOX)-specific inhibitor was fed to flies with ifc-KO photoreceptors. Apocynin-feeding reduced the level of ROS and partially rescued the neurodegeneration caused by loss of Ifc. Furthermore, with clonal analysis, we observe that Rac1 is activated in ifc-KO photoreceptors. Genetically decreasing the level of Rac1 or NOX partially rescued the function of the degenerated ifc-KO photoreceptors. These results indicate that Rac1-NOX pathway mediates ROS production which causes neurodegeneration in ifc-KO photoreceptors. We also used co-immunoprecipitation to detect the physical interaction between Rac1 and Ifc, but the interaction was not observed, indicating non-association or weak interaction between the two proteins. Interestingly, we have observed the colocalization of Ifc and autophagosome in the photoreceptor. With co-immunoprecipitation, we observed the physical interaction of Ifc and autophagosome. Also, Ifc is predicted to have four LC3-interactiong regions (LIRs) and Ifc with four mutated LIRs failed to rescue the lethality of ifc-KO flies, indicating the importance of Ifc to associate with autophagosomes. These results support that ROS and activity-dependent neurodegeneration is generated through Rac1-NOX pathway upon loss of Ifc in photoreceptors, and the ability of Ifc to interact with autophagosomes is critical for their survival.