脂質代謝在老化中扮演很重要的角色，實驗室先前的研究已經證實了大量表現 inaE/dagl‐1 (二醯甘油酯脂解酶) 或是降低rdgA/dgk‐5 (二醯甘油激酶) 這兩個參與磷酸肌醇循環 (phosphoinositide turnover) 的基因表現均會透過減少雷帕黴素標靶 (TOR) 訊號傳遞而延長果蠅與線蟲的壽命。視網膜退化B基因 (rdgB) 擔任磷脂醯肌醇 (phosphatidylinositol, PI) 轉移蛋白，它在果蠅感光受器的磷酸肌醇循環中扮演重要的角色。我們實驗室先前已經對於rdgB是否參與壽命調控進行一些研究，並揭示了在神經中降低rdgB的表現量可以使果蠅的壽命延長，這促使我們想要進一步去探討rdgB對壽命調控的影響。本篇論文中，我們發現降低rdgB的表現量是以神經元特異性的方式來影響果蠅壽命，在表達dVGluT/Pdf的神經元中具有延長的作用而在表達Trh/tim的神經元中具有縮短的作用。我們發現了rdgB突變體長壽的現象跟光的傳導無關。藉由西方點墨法，我們在rdgB突變體中觀察到磷酸化S6激酶 (p-S6K) 的表現量有降低的趨勢，同樣的結果也在透過RNAi降低不同組織像是神經特異性的Appl、眼睛特異性的GMR以及肌肉特異性的24B中rdgB表現量的果蠅中觀察到。在長壽的rdgB突變體中透過餵食雷帕黴素 (rapamycin) 並不會使原本延長的壽命更進一步增加，此現象也在利用RNAi降低神經特異性的Appl中rdgB表現量的果蠅中觀察到，這些數據都表明了rdgB可能是透過雷帕黴素標靶訊號傳遞來調控壽命。此外，在rdgB突變體中也發現了LC3-II表現量提升以及p62表現量下降的現象。綜上所述，這些結果顯示了降低rdgB表現量是以神經元特異性的方式來延長果蠅的壽命，並且是通過減少雷帕黴素標靶訊號傳遞來進行調控。

Lipid metabolism plays an important role in aging. Our previous research has demonstrated that overexpression of inactivation no afterpotential E/diacylglycerol lipase-1 (inaE/dagl‐1, encoding diacylglycerol lipase) or knockdown of retinal degeneration A/diacylglycerol kinase-5 (rdgA/dgk‐5, encoding diacylglycerol kinase), both of which are the members of phosphoinositide (PI) turnover, increases lifespan by reducing target of rapamycin (TOR) signaling in Drosophila and C. elegans. The retinal degeneration B (rdgB) gene encodes phosphatidylinositol (PI) transfer protein which plays a key role in PI turnover of Drosophila photoreceptor. Whether rdgB participates in lifespan regulation has been studied a little in our laboratory. It revealed that lowered rdgB expression in neuron can lead to extended lifespan in Drosophila. Thus, it prompts us to further explore the effect of rdgB on lifespan regulation. Here, we found that knockdown of rdgB affect lifespan in a neuron-specific manner, with the prolonged effects in dVGluT/Pdf-expressing neurons and the shortened effects in Trh/tim-expressing neurons. We found the longevity phenomenon in rdgB mutant is independent of light transduction. By western blotting, we observed reduced phosphorylated-S6K level in rdgB mutant and RNAi knockdown flies by neuron-specific Appl-GAL4, eye-specific GMR-GAL4 and muscle-specific 24B-GAL4. Rapamycin treatment cannot further extend lifespan in long-lived rdgB mutant and knockdown flies by neuron-specific Appl-GAL4. These data suggest that rdgB regulate lifespan possibly via TOR signaling. In addition, the elevated LC3-II and decreased p62 levels were detected in rdgB mutant as well. Taken together, our results indicate that lowered rdgB expression extends lifespan in a neuron-specific manner and via reduced TOR signaling.

Ch.1 中文摘要(i)
Ch.2 Abstract(ii)
Ch.3 致謝(iii)
Ch.4 Content(1)
Ch.5 Introduction(3)
Ch.6 Materials and methods(6)
6-1 Fly husbandry(6)
6-2 Lifespan assay(6)
6-3 Lifespan assay under GeneSwitch Gal4 system(7)
6-4 Electrophysiology(7)
6-5 RNA extraction and Reverse transcription(7)
6-6 Quantitative Real-time PCR (Q-PCR)(8)
6-7 Immunoblot assay(9)
Ch.7 Results(10)
7-1 rdgB mutant exhibits lifespan extension in Drosophila(10)
7-2 Knockdown of rdgB in neuron and eye extend lifespan but shortens lifespan in muscle(10)
7-3 Pan-neuronal knockdown of rdgB in adulthood prolongs lifespan in female flies(11)
7-4 Knockdown of rdgB in specific neural circuits by dVGluT and Pdf-GAL4 prolongs and by Trh and tim-GAL4 shortens lifespan(12)
7-5 Light-regulated lifespan extension is independent of rdgB-mediated longevity in Drosophila(13)
7-6 rdgBB27337 and rdgB knockdown flies driven by Appl, GMR and 24B-GAL4 display lowered p-S6K levels(14)
7-7 Rapamycin treatment does not further extend lifespan in rdgB mutant and RNAi knockdown flies by Appl-GAL4(14)
7-8 rdgBB27337 exhibits enhanced autophagy activity in Drosophila(15)
Ch.8 Discussion(16)
Ch.9 References(20)
Ch.10 Figures and Tables(23)
Ch.11 Supplementary information(68)

Allemand, R., Cohet, Y., and David, J. (1973). Increase in the longevity of adult Drosophila melanogaster kept in permanent darkness. Experimental gerontology 8, 279-283.
Balla, T. (2013). Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiological reviews 93, 1019-1137.
Bjedov, I., Toivonen, J.M., Kerr, F., Slack, C., Jacobson, J., Foley, A., and Partridge, L. (2010). Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. Cell metabolism 11, 35-46.
Boomgarden, A.C., Sagewalker, G.D., Shah, A.C., Haider, S.D., Patel, P., Wheeler, H.E., Dubowy, C.M., and Cavanaugh, D.J. (2019). Chronic circadian misalignment results in reduced longevity and large-scale changes in gene expression in Drosophila. BMC genomics 20, 14.
Burger, J.M., and Promislow, D.E. (2004). Sex-specific effects of interventions that extend fly life span. Science of aging knowledge environment: SAGE KE 2004, pe30-pe30.
Cockcroft, S. (2012). The diverse functions of phosphatidylinositol transfer proteins. In Phosphoinositides and disease (Springer), pp. 185-208.
Cockcroft, S., and Raghu, P. (2016). Topological organisation of the phosphatidylinositol 4, 5-bisphosphate–phospholipase C resynthesis cycle: PITPs bridge the ER–PM gap. Biochemical Journal 473, 4289-4310.
Fontana, L., Partridge, L., and Longo, V.D. (2010). Extending healthy life span—from yeast to humans. science 328, 321-326.
Guarente, L., and Kenyon, C. (2000). Genetic pathways that regulate ageing in model organisms. Nature 408, 255.
Hansen, M., Taubert, S., Crawford, D., Libina, N., Lee, S.J., and Kenyon, C. (2007). Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans. Aging Cell 6, 95-110.
Jung, C.H., Ro, S.-H., Cao, J., Otto, N.M., and Kim, D.-H. (2010). mTOR regulation of autophagy. FEBS Lett 584, 1287-1295.
Katewa, S.D., Akagi, K., Bose, N., Rakshit, K., Camarella, T., Zheng, X., Hall, D., Davis, S., Nelson, C.S., and Brem, R.B. (2016). Peripheral circadian clocks mediate dietary restriction-dependent changes in lifespan and fat metabolism in Drosophila. Cell metabolism 23, 143-154.
Katz, B., and Minke, B. (2009). Drosophila photoreceptors and signaling mechanisms. Frontiers in cellular neuroscience 3, 2.
Kawli, T., Wu, C., and Tan, M.-W. (2010). Systemic and cell intrinsic roles of Gqα signaling in the regulation of innate immunity, oxidative stress, and longevity in Caenorhabditis elegans. Proceedings of the National Academy of Sciences 107, 13788-13793.
Keinan, O., Kedan, A., Gavert, N., Selitrennik, M., Kim, S., Karn, T., Becker, S., and Lev, S. (2014). The lipid-transfer protein Nir2 enhances epithelial-mesenchymal transition and facilitates breast cancer metastasis. J Cell Sci 127, 4740-4749.
Kenyon, C.J. (2010). The genetics of ageing. Nature 464, 504.
Krauß, M., and Haucke, V. (2007). Phosphoinositides: regulators of membrane traffic and protein function. FEBS Lett 581, 2105-2111.
López-Otín, C., Blasco, M.A., Partridge, L., Serrano, M., and Kroemer, G. (2013). The hallmarks of aging. Cell 153, 1194-1217.
Lee, S.B., Kim, S., Lee, J., Park, J., Lee, G., Kim, Y., Kim, J.M., and Chung, J. (2007). ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase. EMBO reports 8, 360-365.
Lin, Y.-H., Chen, Y.-C., Kao, T.-Y., Lin, Y.-C., Hsu, T.-E., Wu, Y.-C., Ja, W.W., Brummel, T.J., Kapahi, P., Yuh, C.-H., et al. (2014). Diacylglycerol lipase regulates lifespan and oxidative stress response by inversely modulating TOR signaling in Drosophila and C. elegans. Aging Cell 13, 755-764.
Liscovitch, M., and Cantley, L.C. (1995). Signal transduction and membrane traffic: the PITP/phosphoinositide connection. Cell 81, 659-662.
Liu, Y., Wang, W., Shui, G., and Huang, X. (2014). CDP-diacylglycerol synthetase coordinates cell growth and fat storage through phosphatidylinositol metabolism and the insulin pathway. PLoS genetics 10, e1004172.
Loewith, R., and Hall, M.N. (2011). Target of rapamycin (TOR) in nutrient signaling and growth control. Genetics 189, 1177-1201.
Magwere, T., Chapman, T., and Partridge, L. (2004). Sex differences in the effect of dietary restriction on life span and mortality rates in female and male Drosophila melanogaster. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 59, B3-B9.
McGuire, S.E., Roman, G., and Davis, R.L. (2004). Gene expression systems in Drosophila: a synthesis of time and space. TRENDS in Genetics 20, 384-391.
Nicholson, L., Singh, G.K., Osterwalder, T., Roman, G.W., Davis, R.L., and Keshishian, H. (2008). Spatial and temporal control of gene expression in Drosophila using the inducible GeneSwitch GAL4 system. I. Screen for larval nervous system drivers. Genetics 178, 215-234.
Ostojic, I., Boll, W., Waterson, M.J., Chan, T., Chandra, R., Pletcher, S.D., and Alcedo, J. (2014). Positive and negative gustatory inputs affect Drosophila lifespan partly in parallel to dFOXO signaling. Proceedings of the National Academy of Sciences 111, 8143-8148.
Paetkau, D.W., Elagin, V.A., Sendi, L.M., and Hyde, D.R. (1999). Isolation and characterization of Drosophila retinal degeneration B suppressors. Genetics 151, 713-724.
Park, J.-H., Park, J.-W., Lee, J.-H., Kim, D.-Y., Hahm, J.-H., and Bae, Y.-S. (2018). Role of phospholipase D in the lifespan of Caenorhabditis elegans. Experimental & molecular medicine 50, 8.
Parkes, T.L., Elia, A.J., Dickinson, D., Hilliker, A.J., Phillips, J.P., and Boulianne, G.L. (1998). Extension of Drosophila lifespan by overexpression of human SOD1 in motorneurons. Nature Genetics 19, 171-174.
Pittendrigh, C.S., and Minis, D.H. (1972). Circadian systems: longevity as a function of circadian resonance in Drosophila melanogaster. Proceedings of the National Academy of Sciences 69, 1537-1539.
Shostal, O., and Moskalev, A. (2013). The genetic mechanisms of the influence of the light regime on the lifespan of Drosophila melanogaster. Frontiers in genetics 3, 325.
Stanfel, M.N., Shamieh, L.S., Kaeberlein, M., and Kennedy, B.K. (2009). The TOR pathway comes of age. Biochimica et Biophysica Acta (BBA)-General Subjects 1790, 1067-1074.
Tóth, M.L., Sigmond, T., Borsos, É., Barna, J., Erdélyi, P., Takács-Vellai, K., Orosz, L., Kovács, A.L., Csikós, G., and Sass, M. (2008). Longevity pathways converge on autophagy genes to regulate life span in Caenorhabditis elegans. Autophagy 4, 330-338.
Trivedi, D., and Padinjat, R. (2007). RdgB proteins: functions in lipid homeostasis and signal transduction. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids 1771, 692-699.
Vilinsky, I., and Johnson, K.G. (2012). Electroretinograms in Drosophila: a robust and genetically accessible electrophysiological system for the undergraduate laboratory. Journal of Undergraduate Neuroscience Education 11, A149.
Wullschleger, S., Loewith, R., and Hall, M.N. (2006). TOR signaling in growth and metabolism. Cell 124, 471-484.
Zid, B.M., Rogers, A.N., Katewa, S.D., Vargas, M.A., Kolipinski, M.C., Lu, T.A., Benzer, S., and Kapahi, P. (2009). 4E-BP extends lifespan upon dietary restriction by enhancing mitochondrial activity in Drosophila. Cell 139, 149-160.