臺灣博碩士論文加值系統

當遭遇到環境壓力時，生物體內有感應的分子機制在不同胞器中產生相對應的保護反應，稱之為胞器內未摺疊蛋白反應 (UPR)。研究也發現當神經系統遭遇到壓力，能藉由神經訊號產生全身性的胞器內未摺疊蛋白反應，顯示此保護機制能夠被神經訊號所調控。當粒線體的呼吸作用受到抑制或是粒線體中蛋白質穩定失衡，會引發粒線體的未摺疊蛋白反應 (UPRmt)。近來的研究顯示神經系統藉由分泌血清素和神經胜肽FLP-2來調控粒線體的未摺疊蛋白反應。在本篇研究中發現，藉由抑制神經系統中秀麗桿狀線蟲Caenorhabditis elegans調控粒線體的聚合蛋白FZO-1/Mitofusin來破壞神經細胞中的粒線體動態平衡，會引發全身性的粒線體的未摺疊蛋白反應和造成腸道的粒線體型態破碎。並發現乙醯膽鹼、麩胺酸、酪胺、神經胜肽都參與在粒線體的未摺疊蛋白反應中，其中來自RIC和RIM神經的酪胺透過受器TYRA-3完成下游的調控。腸道中粒線體型態改變則是受到神經胜肽而非神經傳導物質鎖調控，暗示這兩種生理現象可能是由不同的神經迴路調控。而我們正在研究神經系統動態平衡改變引起的粒線體未摺疊蛋白反應，參與在何種生理狀態調控和其的生理意義。

Tissue-specific stress responses are protective mechanisms against proteotoxic stress and could be regulated in a non-autonomous fashion. Inhibition of mitochondrial respiration or proteostasis triggers systemic mitochondrial unfolded protein response (UPRmt), and recently serotonin and the FLP-2 neuropeptide had been shown to be important for this regulation. Here we report that disrupting mitochondrial dynamics in the neurons, by silencing the mitochondrial fusion gene fzo-1, induced UPRmt and mitochondrial fragmentation in the intestine. Acetylcholine, tyramine, glutamate and neuropeptides were required to mediate non-autonomous UPRmt. Our data suggest that tyramine signals derived from the RIM and RIC neurons target neurons that express the TYRA-3 tyramine receptor. Strikingly, neuropeptides, but not neurotransmitters, are important for non-autonomous regulation of mitochondrial dynamics in non-neural tissues. Consistent with previous studies linking UPRmt and bacterial defense, fzo-1 mutants showed avoidance to bacterial food. We are now exploring the neural mechanisms that link mitochondrial dynamics to non-autonomous UPRmt regulation and pathogen avoidance.

口試委員會審定書 i
ACKNOWLEDGEMENT ii
中文摘要 iv
ABSTRACT v
CONTENTS vi
Chapter 1 INTRODUCTION 1
1.1 FZO-1/Mitofusins regulate mitochondria dynamics 3
1.2 Cell-non-autonomous regulation of UPRmt 4
1.3 Neuroendocrine signaling and systemic UPRmt regulation 5
1.4 Mitochondrial UPRmt is likely a mechanism for pathogen defense 6
1.5 Current Study: Inter-tissue coordination of UPRmt and mitochondrial morphology via neurotransmitters and neuropeptides 8
Chapter 2 MATERIALS and METHODS 9
2.1 C. elegans Strains and Genetics 9
2.2 Feeding RNA Interference 10
2.3 Fluorescence Microscopy and Quantification of UPRmt GFP Signals 10
2.4 Tyramine Treatment 10
2.5 Imaging of Mitochondrial Morphology 11
2.6 Assays for Bacteria-Avoidance Behavior 11
Chapter 3 RESULTS 12
3.1 Temporal requirement of fzo-1 in UPRmt induction 12
3.2 Neuronal fzo-1 loss induces non-autonomous UPRmt induction 12
3.3 UPRmt in neurons are required for non-autonomous UPRmt induction in the intestine 13
3.4 Acetylcholine, tyramine, glutamate are required for triggering systemic UPRmt upon loss of neuronal fzo-1 14
3.5 Loss of fzo-1 in the cholinergic neurons is sufficient for triggering UPRmt in intestine 15
3.6 Tyramine secretion from either RIC or RIM neurons is sufficient for non-autonomous UPRmt induction 15
3.7 Exogeneous tyramine restored non-autonomous UPRmt in the tdc-1 mutant 16
3.8 tyra-3 is the tyramine receptor required for intestinal UPRmt induction upon neuronal fzo-1 loss 16
3.9 Neuropeptides mediate mitochondrial fragmentation in distal tissues upon loss of neuronal fzo-1 17
3.10 Mitochondrial respiration and dynamics in the neurons differentially regulate mitochondrial morphology in non-neuronal tissue 19
3.11 The fzo-1 mutation induces bacteria avoidance behaviors 20
Chapter 4 DISCUSSION 21
4.1 Temporal requirement of cell-autonomous and non-autonomous UPRmt 21
4.2 Neuroendocrine signaling in UPRmt and mitochondrial morphology regulation 22
4.3 The physiological role of systemic UPRmt 24
Chapter 5 FIGURES 26
Figure 1. fzo-1 loss at early larval stages, but not at late larval or adult stages, induced UPRmt. 27
Figure 2. FLP/FRT-based inducible knockdown of fzo-1 in the neurons. 29
Figure 3. Neuronal fzo-1 RNAi at early, but not late larval stages, triggered non-autonomous UPRmt. 31
Figure 4. Neuronal ubl-5 knockdown ameliorates UPRmt induction by neuronal fzo-1 loss 33
Figure 5. Loss of acetylcholine suppresses UPRmt induction by the fzo-1 mutation. 35
Figure 6. Loss of acetylcholine, glutamate or tyramine suppress UPRmt induction by neuronal fzo-1 knockdown. 37
Figure 7. cco-1 knockdown induces cell-autonomous UPRmt in the unc-17 and tdc-1 mutants. 39
Figure 8. Knockdown of fzo-1 in cholinergic neurons is sufficient for systemic UPRmt induction. 41
Figure 9. Cell-specific TDC-1 expression in the tdc-1 mutant and UPRmt. 43
Figure 10. Exogenous tyramine restored UPRmt induction in the tdc-1 mutant. 45
Figure 11. Acute tyramine treatment restored UPRmt induction in tdc-1 mutant. 47
Figure 12. Loss of the tyramine receptor tyra-3 suppressed UPRmt induction in the fzo-1 mutant. 49
Figure 13. Tyramine supplement does not restore UPRmt in the tyra-3 mutant. 51
Figure 14. Mitochondrial fragmentation in non-neuronal tissue caused by neuronal fzo-1 knockdown. 53
Figure 15. Quantification criteria of mitochondrial fragmentation in non-neuronal tissue. 55
Figure 16. Mitochondrial morphology is fragmented in non-neuronal tissue of fzo-1 mutant. 57
Figure 17. Neuronal fzo-1 loss triggers mitochondrial fragmentation in both the wild type and the sid-1 mutant background. 59
Figure 18. The unc-31 mutation blocked mitochondrial fragmentation triggered by neuronal fzo-1 knockdown. 61
Figure 19. Biogenic amine signaling is not involved in mediating mitochondrial fragmentation triggered by neuronal fzo-1 knockdown. 63
Figure 20. Quantification of mitochondrial fragmentation triggered by neuronal fzo-1 knockdown in mutants that lacked biogenic amine signaling. 65
Figure 21. Knockdown of cco-1 in the neurons does not trigger mitochondrial fragmentation in non-neuronal tissue. 67
Figure 22. The fzo-1 mutant showed bacteria avoidance behavior. 69
Figure 23. Model of non-autonomous regulation of mitochondrial stress and morphology by the neurons. 71
Chapter 6 REFERENCE 73

Alkema, M.J., Hunter-Ensor, M., Ringstad, N., and Horvitz, H.R. (2005). Tyramine Functions independently of octopamine in the Caenorhabditis elegans nervous system. Neuron 46, 247-260.
Balch, W.E., Morimoto, R.I., Dillin, A., and Kelly, J.W. (2008). Adapting proteostasis for disease intervention. Science (New York, N.Y.) 319, 916-919.
Berendzen, K.M., Durieux, J., Shao, L.W., Tian, Y., Kim, H.E., Wolff, S., Liu, Y., and Dillin, A. (2016). Neuroendocrine Coordination of Mitochondrial Stress Signaling and Proteostasis. Cell 166, 1553-1563 e1510.
Bos, L.D.J., Sterk, P.J., and Schultz, M.J. (2013). Volatile Metabolites of Pathogens: A Systematic Review. PLOS Pathogens 9, e1003311.
Breckenridge, D.G., Kang, B.H., Kokel, D., Mitani, S., Staehelin, L.A., and Xue, D. (2008). Caenorhabditis elegans drp-1 and fis-2 regulate distinct cell-death execution pathways downstream of ced-3 and independent of ced-9. Molecular cell 31, 586-597.
Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94.
Burkewitz, K., Morantte, I., Weir, H.J., Yeo, R., Zhang, Y., Huynh, F.K., Ilkayeva, O.R., Hirschey, M.D., Grant, A.R., and Mair, W.B. (2015). Neuronal CRTC-1 governs systemic mitochondrial metabolism and lifespan via a catecholamine signal. Cell 160, 842-855.
Chan, D.C. (2012). Fusion and Fission: Interlinked Processes Critical for Mitochondrial Health. Annual Review of Genetics 46, 265-287.
Chang, H.C., Paek, J., and Kim, D.H. (2011). Natural polymorphisms in C. elegans HECW-1 E3 ligase affect pathogen avoidance behaviour. Nature 480, 525-529.
Davis, M.W., Morton, J.J., Carroll, D., and Jorgensen, E.M. (2008). Gene Activation Using FLP Recombinase in C. elegans. PLoS genetics 4, e1000028.
de Brito, O.M., and Scorrano, L. (2008). Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 456, 605-610.
Dillin, A., Hsu, A.L., Arantes-Oliveira, N., Lehrer-Graiwer, J., Hsin, H., Fraser, A.G., Kamath, R.S., Ahringer, J., and Kenyon, C. (2002). Rates of behavior and aging specified by mitochondrial function during development. Science (New York, N.Y.) 298, 2398-2401.
Donnelly, J.L., Clark, C.M., Leifer, A.M., Pirri, J.K., Haburcak, M., Francis, M.M., Samuel, A.D.T., and Alkema, M.J. (2013). Monoaminergic Orchestration of Motor Programs in a Complex C. elegans Behavior. PLOS Biology 11, e1001529.
Douglas, P.M., Baird, N.A., Simic, M.S., Uhlein, S., McCormick, M.A., Wolff, S.C., Kennedy, B.K., and Dillin, A. (2015). Heterotypic Signals from Neural HSF-1 Separate Thermotolerance from Longevity. Cell Rep 12, 1196-1204.
Durieux, J., Wolff, S., and Dillin, A. (2011). The cell-non-autonomous nature of electron transport chain-mediated longevity. Cell 144, 79-91.
Escoll, P., Mondino, S., Rolando, M., and Buchrieser, C. (2016). Targeting of host organelles by pathogenic bacteria: a sophisticated subversion strategy. Nature reviews. Microbiology 14, 5-19.
Eura, Y., Ishihara, N., Yokota, S., and Mihara, K. (2003). Two mitofusin proteins, mammalian homologues of FZO, with distinct functions are both required for mitochondrial fusion. Journal of biochemistry 134, 333-344.
Filadi, R., Greotti, E., Turacchio, G., Luini, A., Pozzan, T., and Pizzo, P. (2015). Mitofusin 2 ablation increases endoplasmic reticulum–mitochondria coupling. Proceedings of the National Academy of Sciences 112, E2174-E2181.
Guo, S., and Kemphues, K.J. (1995). par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81, 611-620.
Jiang, H.C., Hsu, J.M., Yen, C.P., Chao, C.C., Chen, R.H., and Pan, C.L. (2015). Neural activity and CaMKII protect mitochondria from fragmentation in aging Caenorhabditis elegans neurons. Proceedings of the National Academy of Sciences of the United States of America 112, 8768-8773.
Johnson, D., and Nehrke, K. (2010). Mitochondrial fragmentation leads to intracellular acidification in Caenorhabditis elegans and mammalian cells. Molecular biology of the cell 21, 2191-2201.
Kamath, R.S., Martinez-Campos, M., Zipperlen, P., Fraser, A.G., and Ahringer, J. (2001). Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome biology 2, Research0002.
Kirienko, N.V., Ausubel, F.M., and Ruvkun, G. (2015). Mitophagy confers resistance to siderophore-mediated killing by Pseudomonas aeruginosa. Proceedings of the National Academy of Sciences of the United States of America 112, 1821-1826.
Lin, C.-T. (2016). Neural regulation of mitochondrial stress response in Caenorhabditis elegans. (Unpublished: National Taiwan University).
Liu, Y., Samuel, B.S., Breen, P.C., and Ruvkun, G. (2014). Caenorhabditis elegans pathways that surveil and defend mitochondria. Nature 508, 406-410.
Liu, Y., Schweitzer, E.S., Nirenberg, M.J., Pickel, V.M., Evans, C.J., and Edwards, R.H. (1994). Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells. J Cell Biol 127, 1419-1433.
Loer, C.R., JB (2016). The Evidence for Classical Neurotransmitters in Caenorhabditis elegans. in WormAtlas.
Lotze, M.T., Zeh, H.J., Rubartelli, A., Sparvero, L.J., Amoscato, A.A., Washburn, N.R., Devera, M.E., Liang, X., Tor, M., and Billiar, T. (2007). The grateful dead: damage-associated molecular pattern molecules and reduction/oxidation regulate immunity. Immunological reviews 220, 60-81.
Madison, J.M., Nurrish, S., and Kaplan, J.M. (2005). UNC-13 interaction with syntaxin is required for synaptic transmission. Curr Biol 15, 2236-2242.
Meisel, J.D., Panda, O., Mahanti, P., Schroeder, F.C., and Kim, D.H. (2014). Chemosensation of bacterial secondary metabolites modulates neuroendocrine signaling and behavior of C. elegans. Cell 159, 267-280.
Mello, C.C., Kramer, J.M., Stinchcomb, D., and Ambros, V. (1991). Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. The EMBO journal 10, 3959-3970.
Melo, J.A., and Ruvkun, G. (2012). Inactivation of conserved C. elegans genes engages pathogen- and xenobiotic-associated defenses. Cell 149, 452-466.
Merkwirth, C., Jovaisaite, V., Durieux, J., Matilainen, O., Jordan, S.D., Quiros, P.M., Steffen, K.K., Williams, E.G., Mouchiroud, L., Tronnes, S.U., et al. (2016). Two Conserved Histone Demethylases Regulate Mitochondrial Stress-Induced Longevity. Cell 165, 1209-1223.
Naon, D., Zaninello, M., Giacomello, M., Varanita, T., Grespi, F., Lakshminaranayan, S., Serafini, A., Semenzato, M., Herkenne, S., Hernández-Alvarez, M.I., et al. (2016). Critical reappraisal confirms that Mitofusin 2 is an endoplasmic reticulum–mitochondria tether. Proceedings of the National Academy of Sciences 113, 11249-11254.
Nargund, A.M., Fiorese, C.J., Pellegrino, M.W., Deng, P., and Haynes, C.M. (2015). Mitochondrial and nuclear accumulation of the transcription factor ATFS-1 promotes OXPHOS recovery during the UPR(mt). Molecular cell 58, 123-133.
Nargund, A.M., Pellegrino, M.W., Fiorese, C.J., Baker, B.M., and Haynes, C.M. (2012). Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation. Science (New York, N.Y.) 337, 587-590.
Newton, K., and Dixit, V.M. (2012). Signaling in innate immunity and inflammation. Cold Spring Harbor perspectives in biology 4.
Pellegrino, M.W., Nargund, A.M., Kirienko, N.V., Gillis, R., Fiorese, C.J., and Haynes, C.M. (2014). Mitochondrial UPR-regulated innate immunity provides resistance to pathogen infection. Nature 516, 414-417.
Pena, S., Sherman, T., Brookes, P.S., and Nehrke, K. (2016). The Mitochondrial Unfolded Protein Response Protects against Anoxia in Caenorhabditis elegans. PLoS One 11, e0159989.
Pirri, J.K., McPherson, A.D., Donnelly, J.L., Francis, M.M., and Alkema, M.J. (2009). A tyramine-gated chloride channel coordinates distinct motor programs of a Caenorhabditis elegans escape response. Neuron 62, 526-538.
Pradel, E., Zhang, Y., Pujol, N., Matsuyama, T., Bargmann, C.I., and Ewbank, J.J. (2007). Detection and avoidance of a natural product from the pathogenic bacterium Serratia marcescens by Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America 104, 2295-2300.
Prahlad, V., Cornelius, T., and Morimoto, R.I. (2008). Regulation of the cellular heat shock response in Caenorhabditis elegans by thermosensory neurons. Science (New York, N.Y.) 320, 811-814.
Reddy, K.C., Andersen, E.C., Kruglyak, L., and Kim, D.H. (2009). A polymorphism in npr-1 is a behavioral determinant of pathogen susceptibility in C. elegans. Science (New York, N.Y.) 323, 382-384.
Rex, E., Hapiak, V., Hobson, R., Smith, K., Xiao, H., and Komuniecki, R. (2005). TYRA-2 (F01E11.5): a Caenorhabditis elegans tyramine receptor expressed in the MC and NSM pharyngeal neurons. Journal of neurochemistry 94, 181-191.
Runkel, E.D., Liu, S., Baumeister, R., and Schulze, E. (2013). Surveillance-activated defenses block the ROS-induced mitochondrial unfolded protein response. PLoS genetics 9, e1003346.
Schroeder, Elizabeth A., Raimundo, N., and Shadel, Gerald S. Epigenetic Silencing Mediates Mitochondria Stress-Induced Longevity. Cell Metabolism 17, 954-964.
Shao, L.W., Niu, R., and Liu, Y. (2016). Neuropeptide signals cell non-autonomous mitochondrial unfolded protein response. Cell Res 26, 1182-1196.
Speese, S., Petrie, M., Schuske, K., Ailion, M., Ann, K., Iwasaki, K., Jorgensen, E.M., and Martin, T.F. (2007). UNC-31 (CAPS) is required for dense-core vesicle but not synaptic vesicle exocytosis in Caenorhabditis elegans. J Neurosci 27, 6150-6162.
Tatum, M.C., Ooi, F.K., Chikka, M.R., Chauve, L., Martinez-Velazquez, L.A., Steinbusch, H.W., Morimoto, R.I., and Prahlad, V. (2015). Neuronal serotonin release triggers the heat shock response in C. elegans in the absence of temperature increase. Curr Biol 25, 163-174.
Taylor, R.C., and Dillin, A. (2013). XBP-1 is a cell-nonautonomous regulator of stress resistance and longevity. Cell 153, 1435-1447.
Tian, Y., Garcia, G., Bian, Q., Steffen, K.K., Joe, L., Wolff, S., Meyer, B.J., and Dillin, A. (2016). Mitochondrial Stress Induces Chromatin Reorganization to Promote Longevity and UPR(mt). Cell 165, 1197-1208.
Wragg, R.T., Hapiak, V., Miller, S.B., Harris, G.P., Gray, J., Komuniecki, P.R., and Komuniecki, R.W. (2007). Tyramine and Octopamine Independently Inhibit Serotonin-Stimulated Aversive Behaviors in Caenorhabditis elegans through Two Novel Amine Receptors. The Journal of Neuroscience 27, 13402.
Yoneda, T., Benedetti, C., Urano, F., Clark, S.G., Harding, H.P., and Ron, D. (2004). Compartment-specific perturbation of protein handling activates genes encoding mitochondrial chaperones. Journal of cell science 117, 4055-4066.
Zhang, Y., Lu, H., and Bargmann, C.I. (2005). Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans. Nature 438, 179-184.
Zuchner, S., Mersiyanova, I.V., Muglia, M., Bissar-Tadmouri, N., Rochelle, J., Dadali, E.L., Zappia, M., Nelis, E., Patitucci, A., Senderek, J., et al. (2004). Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A. Nature genetics 36, 449-451.