在神經裡，分子馬達蛋白經由體細胞運送突觸小泡到突觸的過程中，微管(MT)扮演一個重要的角色。我們對於突觸傳遞與神經貨物的搬運已有相當的了解，改變突觸傳遞會直接影響神經軸突的運輸。然而，我們對這些變化會如何更改MT上的轉譯後修飾(PTM)並不清楚。在這項研究中，我們調查了不同突變的秀麗桿線蟲，(帶有各類的突觸傳遞缺陷)，是如何影響軸突的微管蛋白密碼。我們使用了七種突變蟲，分別為dat-1(ok157), eat-4(ky5), unc17(e245), unc25(e156), tbh-1(n3247), tdc-1(ok914), tph-1(mg280)。利用廣泛的技術，例如分析軸突馬達蛋白與貨物的運動、以及西方墨點法(了解PTM表現量)、蟲體免疫染色(了解PTM在MT上的修飾程度)。
從我們的研究結果顯示，這些突觸傳遞缺陷的線蟲，其神經主要的馬達蛋白UNC-104明顯地被影響運動模式，此外，unc17(e245)突變蟲又有顯著乙酰化和酪胺酸化的增加。由於這些結果，我們選擇unc17(e245)作為我們進一步的研究方向，更深入了解UNC-17(編碼囊泡乙酰膽鹼傳輸蛋白，VAChT)如何影響微管PTM以及軸突運輸的深層機制。

Microtubules (MTs) play an important role in trafficking of synaptic vesicles transported by molecular motor proteins from the cell body to the synapse. The relation between neuronal cargo transport and synaptic transmission has been well documented. Changes in synaptic transmission has been shown to directly affect axonal transport, however, little knowledge exists on how these changes affect post-translational modification (PTM) of MTs which in turn alters neuronal transport. In this study, we investigated how C. elegans animals, carrying various mutations that cause various types of synaptic transmission defects, affect the “tubulin code” in axons.
In this study, we employed seven mutant strains with different synaptic transmission defects namely, dat-1(ok157), eat-4(ky5), unc17(e245), unc25(e156), tbh-1(n3247), tdc-1(ok914) and, tph-1(mg280), respectively. We then used a broad range of techniques such as analysis of motor and cargo motility in axons as well as whole worm immunostaining (to understand changes in PTM of axonal microtubules) as well as Western blot analysis (to understand whether PTM enzymes may be up or down regulated).
From our results, it is evident that the major transporter of synaptic vesicles UNC-104/KIF1A (a neuron specific kinesin-3) is largely affected in these synaptic transmission defective worms. Moreover, we found that microtubule acetylation as well as tyrosination significantly changed in unc-17(e245) mutants. Based on these results, future experiments are suggested to focus on investigating the deeper mechanisms on how UNC-17 (encoding for a vesicular acetylcholine transporter, VAChT) affects both PTM of microtubules as well as axonal transport.

References
1. Millecamps, S. and J.P. Julien, Axonal transport deficits and neurodegenerative diseases. Nat Rev Neurosci, 2013. 14(3): p. 161-76.
2. Martin-Cofreces, N.B. and F. Sanchez-Madrid, Sailing to and Docking at the Immune Synapse: Role of Tubulin Dynamics and Molecular Motors. Front Immunol, 2018. 9: p. 1174.
3. Kumar, J., et al., The Caenorhabditis elegans Kinesin-3 motor UNC-104/KIF1A is degraded upon loss of specific binding to cargo. PLoS Genet, 2010. 6(11): p. e1001200.
4. Zhang, Y.V., et al., The KIF1A homolog Unc-104 is important for spontaneous release, postsynaptic density maturation and perisynaptic scaffold organization. Sci Rep, 2017. 7: p. 38172.
5. Hirokawa, N., S. Niwa, and Y. Tanaka, Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease. Neuron, 2010. 68(4): p. 610-38.
6. Kern, J.V., et al., The kinesin-3, unc-104 regulates dendrite morphogenesis and synaptic development in Drosophila. Genetics, 2013. 195(1): p. 59-72.
7. Yonekawa, Y., et al., Defect in synaptic vesicle precursor transport and neuronal cell death in KIF1A motor protein-deficient mice. J Cell Biol, 1998. 141(2): p. 431-41.
8. Hall, D.H. and E.M. Hedgecock, Kinesin-related gene unc-104 is required for axonal transport of synaptic vesicle in C. elegans. 1991.
9. Hirokawa, N. and R. Takemura, Molecular motors and mechanisms of directional transport in neurons. Nat Rev Neurosci, 2005. 6(3): p. 201-14.
10. Maeder, C.I., et al., In vivo neuron-wide analysis of synaptic vesicle precursor trafficking. Traffic, 2014. 15(3): p. 273-91.
11. Fischer von Mollard, G., et al., rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles. Proc Natl Acad Sci U S A, 1990. 87(5): p. 1988-92.
12. Nonet, M.L., Caenorhabditis elegans rab-3 mutant synapses exhibit impaired function and are partially depleted of vesicles. 1997.
13. dhof, T.C.S., Function of Rab3 GDP–GTP Exchange. 1997.
14. Tanaka, M., Role of Rab3 GDP-GTP Exchange Protein in Synaptic Vesicle Trafficking at the Mouse Neuromuscular Junction. 2001.
15. Horgan, C.P. and M.W. McCaffrey, Rab GTPases and microtubule motors. Biochem Soc Trans, 2011. 39(5): p. 1202-6.
16. Goldstein, A.Y., X. Wang, and T.L. Schwarz, Axonal transport and the delivery of pre-synaptic components. Curr Opin Neurobiol, 2008. 18(5): p. 495-503.
17. Fischer von Mollard, G., T.C. Sudhof, and R. Jahn, A small GTP-binding protein dissociates from synaptic vesicles during exocytosis. Nature, 1991. 349(6304): p. 79-81.
18. Lovinger, D.M., Communication networks in the brain: neurons, receptors, neurotransmitters, and alcohol. Alcohol Res Health, 2008. 31(3): p. 196-214.
19. Sudhof, T.C. and R.C. Malenka, Understanding synapses: past, present, and future. Neuron, 2008. 60(3): p. 469-76.
20. Wilcox, K.S., J. Buchhalter, and M.A. Dichter, Properties of inhibitory and excitatory synapses between hippocampal neurons in very low density cultures. Synapse, 1994. 18(2): p. 128-51.
21. Matteoli, M. and P. De Camilli, Molecular mechanisms in neurotransmitter release. Curr Opin Neurobiol, 1991. 1(1): p. 91-7.
22. Fon, E.A. and R.H. Edwards, Molecular mechanisms of neurotransmitter release. Muscle Nerve, 2001. 24(5): p. 581-601.
23. Gasnier, B., The loading of neurotransmitters into synaptic vesicles. Biochimie, 2000. 82(4): p. 327-37.
24. Blakely, R.D. and R.H. Edwards, Vesicular and plasma membrane transporters for neurotransmitters. Cold Spring Harb Perspect Biol, 2012. 4(2).
25. Verhey, K.J. and J. Gaertig, The tubulin code. Cell Cycle, 2007. 6(17): p. 2152-60.
26. Shida, T., et al., The major alpha-tubulin K40 acetyltransferase alphaTAT1 promotes rapid ciliogenesis and efficient mechanosensation. Proc Natl Acad Sci U S A, 2010. 107(50): p. 21517-22.
27. Westermann, S. and K. Weber, Post-translational modifications regulate microtubule function. Nat Rev Mol Cell Biol, 2003. 4(12): p. 938-47.
28. Song, Y. and S.T. Brady, Post-translational modifications of tubulin: pathways to functional diversity of microtubules. Trends Cell Biol, 2015. 25(3): p. 125-36.
29. JW, H., Tubulin modifications and their cellular functions. 2008.
30. Sirajuddin, M., L.M. Rice, and R.D. Vale, Regulation of microtubule motors by tubulin isotypes and post-translational modifications. Nat Cell Biol, 2014. 16(4): p. 335-44.
31. Janke, C. and J.C. Bulinski, Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions. Nat Rev Mol Cell Biol, 2011. 12(12): p. 773-86.
32. Alexander Palazzo, B.A., Gregg G. Gundersen, Tubulin acetylation and cell motility.pdf. 2003.
33. Post-translational modifications of tubulin.
34. Perdiz, D., et al., The ins and outs of tubulin acetylation: more than just a post-translational modification? Cell Signal, 2011. 23(5): p. 763-71.
35. Reed, N.A., et al., Microtubule acetylation promotes kinesin-1 binding and transport. Curr Biol, 2006. 16(21): p. 2166-72.
36. Peris, L., et al., Motor-dependent microtubule disassembly driven by tubulin tyrosination. J Cell Biol, 2009. 185(7): p. 1159-66.
37. Dunn, S., et al., Differential trafficking of Kif5c on tyrosinated and detyrosinated microtubules in live cells. J Cell Sci, 2008. 121(Pt 7): p. 1085-95.
38. Pirri, J.K. and M.J. Alkema, The neuroethology of C. elegans escape. Curr Opin Neurobiol, 2012. 22(2): p. 187-93.
39. Ziv, N.E. and C.C. Garner, Cellular and molecular mechanisms of presynaptic assembly. Nat Rev Neurosci, 2004. 5(5): p. 385-99.
40. Schlager, M.A. and C.C. Hoogenraad, Basic mechanisms for recognition and transport of synaptic cargos. Mol Brain, 2009. 2: p. 25.
41. Brenner, S., The genetics of Caenorhabditis elegans. Genetics, 1974. 77(1): p. 71-94.
42. Nass, R., et al., Neurotoxin-induced degeneration of dopamine neurons in Caenorhabditis elegans. Proc Natl Acad Sci U S A, 2002. 99(5): p. 3264-9.
43. Avery, L., The genetics of feeding in Caenorhabditis elegans. Genetics, 1993. 133(4): p. 897-917.
44. Zhu, H., et al., Analysis of point mutants in the Caenorhabditis elegans vesicular acetylcholine transporter reveals domains involved in substrate translocation. J Biol Chem, 2001. 276(45): p. 41580-7.
45. de Castro, B.M., et al., The vesicular acetylcholine transporter is required for neuromuscular development and function. Mol Cell Biol, 2009. 29(19): p. 5238-50.
46. McIntire, S.L., et al., The GABAergic nervous system of Caenorhabditis elegans. Nature, 1993. 364(6435): p. 337-41.
47. Jin, Y., et al., The Caenorhabditis elegans gene unc-25 encodes glutamic acid decarboxylase and is required for synaptic transmission but not synaptic development. J Neurosci, 1999. 19(2): p. 539-48.
48. Alkema, M.J., et al., Tyramine Functions independently of octopamine in the Caenorhabditis elegans nervous system. Neuron, 2005. 46(2): p. 247-60.
49. Sze, J.Y., et al., Food and metabolic signalling defects in a Caenorhabditis elegans serotonin-synthesis mutant. Nature, 2000. 403(6769): p. 560-4.
50. Tien, N.W., et al., Tau/PTL-1 associates with kinesin-3 KIF1A/UNC-104 and affects the motor's motility characteristics in C. elegans neurons. Neurobiol Dis, 2011. 43(2): p. 495-506.
51. Mello, C. and A. Fire, DNA transformation. Methods Cell Biol, 1995. 48: p. 451-82.
52. Kamath, R.S., et al., Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol, 2001. 2(1): p. RESEARCH0002.
53. Anderson, J.L., L.T. Morran, and P.C. Phillips, Outcrossing and the maintenance of males within C. elegans populations. J Hered, 2010. 101 Suppl 1: p. S62-74.
54. Mahmood, T. and P.C. Yang, Western blot: technique, theory, and trouble shooting. N Am J Med Sci, 2012. 4(9): p. 429-34.
55. Duerr, J.S., Immunohistochemistry. WormBook, 2006: p. 1-61.
56. Konishi, Y. and M. Setou, Tubulin tyrosination navigates the kinesin-1 motor domain to axons. Nat Neurosci, 2009. 12(5): p. 559-67.
57. Wagner, O.I., et al., Synaptic scaffolding protein SYD-2 clusters and activates kinesin-3 UNC-104 in C. elegans. Proc Natl Acad Sci U S A, 2009. 106(46): p. 19605-10.
58. Ikegami, K., et al., Loss of alpha-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function. Proc Natl Acad Sci U S A, 2007. 104(9): p. 3213-8.
59. Yogev, S., et al., Local inhibition of microtubule dynamics by dynein is required for neuronal cargo distribution. Nat Commun, 2017. 8: p. 15063.
60. Francis, P.T., The interplay of neurotransmitters in Alzheimer's disease. CNS Spectr, 2005. 10(11 Suppl 18): p. 6-9.
61. Factor, S.A., W.M. McDonald, and F.C. Goldstein, The role of neurotransmitters in the development of Parkinson's disease-related psychosis. Eur J Neurol, 2017. 24(10): p. 1244-1254.
62. Garcia-Alloza, M., et al., Cholinergic-serotonergic imbalance contributes to cognitive and behavioral symptoms in Alzheimer's disease. Neuropsychologia, 2005. 43(3): p. 442-9.
63. Richmond, J.E. and E.M. Jorgensen, One GABA and two acetylcholine receptors function at the C-elegans neuromuscular junction. Nature Neuroscience, 1999. 2(9): p. 791-797.
64. Vasil’eva, N.A. and A.S. Pivovarov, Microtubule Motor Proteins and the Mechanisms of Synaptic Plasticity. Neuroscience and Behavioral Physiology, 2017. 47(5): p. 585-594.
65. Maas, C., et al., Synaptic activation modifies microtubules underlying transport of postsynaptic cargo. Proc Natl Acad Sci U S A, 2009. 106(21): p. 8731-6.
66. Wloga, D., E. Joachimiak, and H. Fabczak, Tubulin Post-Translational Modifications and Microtubule Dynamics. Int J Mol Sci, 2017. 18(10).
67. Szczypka, M.S., et al., Feeding behavior in dopamine-deficient mice. Proc Natl Acad Sci U S A, 1999. 96(21): p. 12138-43.
68. Greenwood, T.A., et al., Identification of additional variants within the human dopamine transporter gene provides further evidence for an association with bipolar disorder in two independent samples. Mol Psychiatry, 2006. 11(2): p. 125-33, 115.
69. Kume, K., et al., Dopamine is a regulator of arousal in the fruit fly. J Neurosci, 2005. 25(32): p. 7377-84.
70. Singh, K., et al., Deep conservation of genes required for both Drosphila melanogaster and Caenorhabditis elegans sleep includes a role for dopaminergic signaling. Sleep, 2014. 37(9): p. 1439-51.
71. Barros, A.G., et al., Dopamine signaling regulates fat content through beta-oxidation in Caenorhabditis elegans. PLoS One, 2014. 9(1): p. e85874.
72. Maricq, A.V., et al., Mechanosensory signalling in C. elegans mediated by the GLR-1 glutamate receptor. Nature, 1995. 378(6552): p. 78-81.
73. Hart, A.C., S. Sims, and J.M. Kaplan, Synaptic code for sensory modalities revealed by C. elegans GLR-1 glutamate receptor. Nature, 1995. 378(6552): p. 82-5.
74. Lee, R.Y., et al., EAT-4, a homolog of a mammalian sodium-dependent inorganic phosphate cotransporter, is necessary for glutamatergic neurotransmission in caenorhabditis elegans. J Neurosci, 1999. 19(1): p. 159-67.
75. Dent, J.A., M.W. Davis, and L. Avery, avr-15 encodes a chloride channel subunit that mediates inhibitory glutamatergic neurotransmission and ivermectin sensitivity in Caenorhabditis elegans. EMBO J, 1997. 16(19): p. 5867-79.
76. McIntire, S.L., E. Jorgensen, and H.R. Horvitz, Genes required for GABA function in Caenorhabditis elegans. Nature, 1993. 364(6435): p. 334-7.
77. Rex, E. and R.W. Komuniecki, Characterization of a tyramine receptor from Caenorhabditis elegans. J Neurochem, 2002. 82(6): p. 1352-9.
78. Monastirioti, M., C.E. Linn, Jr., and K. White, Characterization of Drosophila tyramine beta-hydroxylase gene and isolation of mutant flies lacking octopamine. J Neurosci, 1996. 16(12): p. 3900-11.
79. Suo, S., Y. Kimura, and H.H. Van Tol, Starvation induces cAMP response element-binding protein-dependent gene expression through octopamine-Gq signaling in Caenorhabditis elegans. J Neurosci, 2006. 26(40): p. 10082-90.
80. Greer, E.R., et al., Neural and molecular dissection of a C. elegans sensory circuit that regulates fat and feeding. Cell Metab, 2008. 8(2): p. 118-31.
81. Huser, A., et al., The serotonergic central nervous system of the Drosophila larva: anatomy and behavioral function. PLoS One, 2012. 7(10): p. e47518.
82. Pocock, R. and O. Hobert, Hypoxia activates a latent circuit for processing gustatory information in C. elegans. Nat Neurosci, 2010. 13(5): p. 610-4.
83. Clark, S.G. and C. Chiu, C. elegans ZAG-1, a Zn-finger-homeodomain protein, regulates axonal development and neuronal differentiation. Development, 2003. 130(16): p. 3781-94.
84. Sandoval, G.M., et al., A genetic interaction between the vesicular acetylcholine transporter VAChT/UNC-17 and synaptobrevin/SNB-1 in C. elegans. Nat Neurosci, 2006. 9(5): p. 599-601.
85. BRENNER, S., THE GENETICS OF CAENORHABDZTZS ELEGANS. 1973.
86. RAND, J.B., Choline acetyltransferase-deficient mutants of the nematode Caenorhabditis elegans. 1983.
87. Aixa Alfonso, K.G., Janet S. Duerr, He-Ping Han, and J.B. Randt, The Caenorhabditis elegans unc- 17 Gene- A Putative Vesicular Acetylcholine Transporter.full. 1993.
88. Mathews, E.A., et al., Genetic interactions between UNC-17/VAChT and a novel transmembrane protein in Caenorhabditis elegans. Genetics, 2012. 192(4): p. 1315-25.
89. Rand, J.B., Genetic Analysis of the cha-l-unc-17 Gene Complex in Caenorhabditis. 1989.
90. Pereira, L., et al., A cellular and regulatory map of the cholinergic nervous system of C. elegans. Elife, 2015. 4.
91. Nguyen, M., Caenorhabditis elegans mutants resistant to inhibitors of acetylcholinesterase. 1995.
92. Mufson, E.J., et al., Cholinergic system during the progression of Alzheimer's disease: therapeutic implications. Expert Rev Neurother, 2008. 8(11): p. 1703-18.
93. Ferreira-Vieira, T.H., et al., Alzheimer's disease: Targeting the Cholinergic System. Curr Neuropharmacol, 2016. 14(1): p. 101-15.
94. Yogev, S., et al., Microtubule Organization Determines Axonal Transport Dynamics. Neuron, 2016. 92(2): p. 449-460.
95. Kaul, N., V. Soppina, and K.J. Verhey, Effects of alpha-tubulin K40 acetylation and detyrosination on kinesin-1 motility in a purified system. Biophys J, 2014. 106(12): p. 2636-43.
96. Liao, G., Kinesin Is a Candidate for Cross-bridging Microtubules and Intermediate Filaments. 1998.
97. Walter, W.J., et al., Tubulin acetylation alone does not affect kinesin-1 velocity and run length in vitro. PLoS One, 2012. 7(8): p. e42218.
98. Magiera, M.M. and C. Janke, Post-translational modifications of tubulin. Curr Biol, 2014. 24(9): p. R351-4.
99. Gross, S.P., Hither and yon: a review of bi-directional microtubule-based transport. Phys Biol, 2004. 1(1-2): p. R1-11.
100. Tanenbaum, M.E., A. Akhmanova, and R.H. Medema, Bi-directional transport of the nucleus by dynein and kinesin-1. Commun Integr Biol, 2011. 4(1): p. 21-5.
101. Schuster, M., et al., Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array. Mol Biol Cell, 2011. 22(19): p. 3645-57.
102. Alper, J.D., et al., The motility of axonemal dynein is regulated by the tubulin code. Biophys J, 2014. 107(12): p. 2872-2880.
103. Kalebic, N., et al., alphaTAT1 is the major alpha-tubulin acetyltransferase in mice. Nat Commun, 2013. 4: p. 1962.
104. McKenney, R.J., et al., Tyrosination of alpha-tubulin controls the initiation of processive dynein-dynactin motility. EMBO J, 2016. 35(11): p. 1175-85.