在本研究主要利用whole-cell patch clamp的電生理技術，探討神經性蛇毒notexin在爪蟾 (Xenopus Laevis) 的運動神經─肌細胞突觸上的作用機轉。Notexin造成運動神經─肌細胞突觸的自發性突觸電流增強並和濃度有正相關的現象。而此自發性突觸電流增強的現象可藉置換細胞培養液為無鈣的Ringer溶液或加入細胞內鈣離子螯合劑BAPTA所抑制，顯示出notexin造成突觸電流增強的現象需要大量胞外鈣離子流入細胞內。SKF96365是廣效性的TRP channel抑制劑，在我們的動物模式上能有效抑制notexin造成的突觸電流增強現象，但L-type電壓敏感型鈣離子通道抑制劑nifedipine及verapamil則沒有抑制的效果。此外，藉由與TRPC channel具有專一性的morpholino降低TRPC 通道蛋白表現時，也能抑制notexin造成的電流增強現象，因此能更進一步瞭解TRPC channel是notexin作用時細胞外鈣離子流入細胞內的主要通道。在細胞培養中事先加入Ca2+-ATPase抑制劑thapsigargin或CPA使細胞內鈣離子貯存池排空，在這種情況下也能明顯地抑制notexin作用造成的突觸電流增強效果。事先處理具有細胞膜通透力的inositol 1,4,5-triphosphate (IP3) 抑制劑Xec，也能有效壓制notexin造成的突觸電流增強作用，並且notexin的作用也能被phospholipase C (PLC) 抑制劑U73122所抑制。因此總合上述的結果，notexin引起細胞內鈣離子貯存池釋出鈣離子並進一步造成鈣離子貯存池排空所調控的鈣離子通道開啟，細胞外鈣離子藉由開啟的TRPC通道流入細胞內，並且經由PLC訊息傳遞路徑造成突觸前自發性訊息傳遞物質大量釋放。
藉由化學物質phenylglyoxal對notexin作的化學修飾物可以明顯地降低notexin造成的增強突觸電流，事先處理PLA2抑制劑aristolochic acid和glycyhirrzin也可抑制notexin的作用，由此可知，PLA2活性參與notexin造成突觸電流增強的作用。先前的研究指出，在老鼠小腦神經細胞培養中發現，由神經性蛇毒分解細胞膜後的產物：myristoyl lysophosphatidylcholine (mLPC) 及oleic acid (OA) 混合物累積會促進突觸泡和細胞膜融合，進而造成大量的神經傳遞物質釋放，並且造成神經細胞神經突 (neurite) 膨大的現象。但在我們的電生理記錄及同步的影像記錄分析結果發現：加入低濃度notexin後造成神經突膨大現象的發生時間明顯早於大量神經傳遞物質釋放所發生的時間。除此之外，在電生理記錄的結果：單獨給予mLPC、OA或者mLPC及OA的混合物皆無法模擬神經性蛇毒造成突觸大量神經傳遞物質釋放的現象。PLA2活性衰減的化學性修飾notexin (notexin-80)造成的神經傳遞物質釋放刺激相對的比notexin造成的影響低，但其對神經突外觀的變化和notexin相比並無明顯的差異。此外，特定序列修飾且具有完整PLA2活性的notexin (PLP-notexin)，則是明顯降低了對神經突造成退化性現象的作用。因此，根據我們的實驗結果，認為notexin造成的神經外觀型態改變及突觸大量神經傳遞物質釋放兩個事件是經由不同的分子機轉所調控。

The mechanism of action of notexin in the facilitation of spontaneous transmitter release at neuromuscular synapse was investigated in Xenopus cell culture by using whole-cell patch clamp recording. Exposure of the culture to notexin dose-dependently enhanced the frequency of spontaneous synaptic currents (SSCs). Either buffering of intracellular Ca2+ rise by BAPTA or replacing culture medium with Ca2+-free Ringer’s solution effectively hampered notexin effect, suggesting Ca2+ influx is requisite for this facilitation. Pretreatment of the cultures with a TRP channel inhibitor SKF96365, instead of voltage-dependent L-type Ca2+ channel blockers nifedipine or verapamil, significantly abolished the SSC facilitating effect of notexin. Furthermore, knockdown the expression of TRPC channels by TRPC-specific morpholino abruptly abolished notexin effect, suggesting that TRPC channel is the major entranceway of extracellular Ca2+. The notexin-enhanced SSC frequency was also obviously reduced under intracellular Ca2+ store depletion by pretreatment of the cultures with pharmacological Ca2+-ATPase inhibitor thapsigargin or CPA. Bath application of membrane-permeable inositol 1,4,5-triphosphate (IP3) inhibitor, XeC, effectively occluded the facilitation of SSC frequency elicited by notexin. Furthermore, notexin-induced SSC frequency facilitation was blocked in the presence of phospholipase C (PLC) inhibitor, U73122. Taken collectively, these results suggest that notexin elicits a Ca2+ release from the IP3-sensitive intracellular Ca2+ store which resulted in further store depletion-operated Ca2+ influx through membrane TRPC channels of the presynaptic nerve terminal. This is done via PLC signaling cascade, leading to an enhancement of spontaneous transmitter release.
Notexin-induced synaptic facilitation is potentially reduced while structural modification with phenylglyoxal. In addition, bath application of PLA2 inhibitor either aristolochic acid or glycyhirrzin effectively abolished notexin effect, suggesting the PLA2 activity is involved in notexin-induced SSC frequency facilitation. Previously it has been suggested local accumulation of PLA2-induced lipid metabolites myristoyl lysophosphatidylcholine (mLPC) and oleic acid (OA) promotes the fusion of the hemifused synaptic vesicles with plasma membrane, hence facilitating the neurotransmitter release in cultured cerebella granular neurons and thus resulted in bulges formation along the neurite. Our real-time morphometric analysis and synaptic activity assays showed that bulges formation along the neurite appeared significantly earlier and was induced at lower notexin concentrations than synaptic activity facilitation. Bath application of either mLPC, OA alone or their mixtures failed to mimic the notexin-induced facilitation in spontaneous transmitter release. Attenuation of PLA2 activity by chemical modification (notexin-80) resulted in correlated decrease of notexin-induced synaptic facilitation but not the degenerative morphological sign. Moreover, PLP-notexin, a site-specific modification of notexin with full intact PLA2 activity, shows significant loss the ability of notexin-induced neurite degeneration. Overall, results from our studies suggest the morphologic changes and synaptic facilitating effect induced by notexin are resulted from different cellular mechanisms.

縮 寫 表 ............................................ 1
中文摘要 ............................................ 2
英文摘要 ............................................ 5
緒 論 ............................................ 7
實驗材料 ............................................ 17
實驗方法 ............................................ 22
1. 電生理記錄方式 .......................... 22
2. 實驗數據之統計分析....................... 23
3. 實驗用試劑及供應者 ..................... 24
實驗結果 ............................................ 25
討 論 ............................................ 44
參考文獻 ............................................ 50
圖 表 ............................................ 55

Anderson, M.J., Cohen, M.W., and Zorychta, E. (1977). Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells. J Physiol 268, 731-756.

Bevers, E.M., Comfurius, P., Dekkers, D.W., and Zwaal, R.F. (1999). Lipid translocation across the plasma membrane of mammalian cells. Biochim Biophys Acta 1439, 317-330.

Bobanovic, L.K., Laine, M., Petersen, C.C., Bennett, D.L., Berridge, M.J., Lipp, P., Ripley, S.J., and Bootman, M.D. (1999). Molecular cloning and immunolocalization of a novel vertebrate trp homologue from Xenopus. Biochem J 340 ( Pt 3), 593-599.

Chang, L.S. (1996). Chemical modification of notexin from Notechis scutatus scutatus (Australian tiger snake) venom with pyridoxal-5'-phosphate. J Protein Chem 15, 473-480.

Chang, L.S., Wu, P.F., Liou, J.C., Chiang-Lin, W.H., and Yang, C.C. (2004). Chemical modification of arginine residues of Notechis scutatus scutatus notexin. Toxicon 44, 491-497.

Chapman, E.R. (2002). Synaptotagmin: a Ca(2+) sensor that triggers exocytosis? Nat Rev Mol Cell Biol 3, 498-508.

Cheng, Y.C., Wang, J.J., and Chang, L.S. (2008). B chain is a functional subunit of beta-bungarotoxin for inducing apoptotic death of human neuroblastoma SK-N-SH cells. Toxicon 51, 304-315.

Chernomordik, L.V., Leikina, E., Frolov, V., Bronk, P., and Zimmerberg, J. (1997). An early stage of membrane fusion mediated by the low pH conformation of influenza hemagglutinin depends upon membrane lipids. J Cell Biol 136, 81-93.

Davletov, B., Connell, E., and Darios, F. (2007). Regulation of SNARE fusion machinery by fatty acids. Cell Mol Life Sci 64, 1597-1608.

Eder, A.M., Sasagawa, T., Mao, M., Aoki, J., and Mills, G.B. (2000). Constitutive and lysophosphatidic acid (LPA)-induced LPA production: role of phospholipase D and phospholipase A2. Clin Cancer Res 6, 2482-2491.

Fathi, H.B., Rowan, E.G., and Harvey, A.L. (2001). The facilitatory actions of snake venom phospholipase A(2) neurotoxins at the neuromuscular junction are not mediated through voltage-gated K(+) channels. Toxicon 39, 1871-1882.

Fiskum, G. (2000). Mitochondrial participation in ischemic and traumatic neural cell death. J Neurotrauma 17, 843-855.

Fletcher, J.E., and Jiang, M.S. (1995). Presynaptically acting snake venom phospholipase A2 enzymes attack unique substrates. Toxicon 33, 1565-1576.

Gaits, F., Fourcade, O., Le Balle, F., Gueguen, G., Gaige, B., Gassama-Diagne, A., Fauvel, J., Salles, J.P., Mauco, G., Simon, M.F., and Chap, H. (1997).
Lysophosphatidic acid as a phospholipid mediator: pathways of synthesis. FEBS Lett 410, 54-58.

Golfman, L.S., Haughey, N.J., Wong, J.T., Jiang, J.Y., Lee, D., Geiger, J.D., and Choy, P.C. (1999). Lysophosphatidylcholine induces arachidonic acid release and calcium overload in cardiac myoblastic H9c2 cells. J Lipid Res 40, 1818-1826.

Halpert, J., and Eaker, D. (1975). Amino acid sequence of a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake). J Biol Chem 250, 6990-6997.

Hamill, O.P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F.J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391, 85-100.

Harish, O.E., and Poo, M.M. (1992). Retrograde modulation at developing neuromuscular synapses: involvement of G protein and arachidonic acid cascade. Neuron 9, 1201-1209.

Harris, J.B., Grubb, B.D., Maltin, C.A., and Dixon, R. (2000). The neurotoxicity of the venom phospholipases A(2), notexin and taipoxin. Exp Neurol 161, 517-526.

Ichas, F., and Mazat, J.P. (1998). From calcium signaling to cell death: two conformations for the mitochondrial permeability transition pore. Switching from low- to high-conductance state. Biochim Biophys Acta 1366, 33-50.

Ikegami, K., Kato, S., and Koike, T. (2004). N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) suppresses neuritic degeneration caused by different experimental paradigms including in vitro Wallerian degeneration. Brain Res 1030, 81-93.

Kao, P.H., Lin, S.R., and Chang, L.S. (2007). Phospholipase A2 activity-independent membrane-damaging effect of notexin. Toxicon 50, 952-959.

Koike, T., Yang, Y., Suzuki, K., and Zheng, X. (2008). Axon & dendrite degeneration: its mechanisms and protective experimental paradigms. Neurochem Int 52, 751-760.

MacInnis, B.L., and Campenot, R.B. (2005). Regulation of Wallerian degeneration and nerve growth factor withdrawal-induced pruning of axons of sympathetic neurons by the proteasome and the MEK/Erk pathway. Mol Cell Neurosci 28, 430-439.

Nacira Tabti, J.A., and Mu-ming Poo (1998). Culturing spinal neurons and musle cells from Xenopus embryos. In Culturing nerve cells G. Banker, and K. Goslin, eds. (Cambridge, Mass.: MIT Press), pp. xii, 666 p., 611 p. of plates.

Rapuano, B.E., Yang, C.C., and Rosenberg, P. (1986). The relationship between high-affinity noncatalytic binding of snake venom phospholipases A2 to brain synaptic plasma membranes and their central lethal potencies. Biochim Biophys Acta 856, 457-470.

Rigoni, M., Caccin, P., Gschmeissner, S., Koster, G., Postle, A.D., Rossetto, O., Schiavo, G., and Montecucco, C. (2005). Equivalent effects of snake PLA2 neurotoxins and lysophospholipid-fatty acid mixtures. Science 310, 1678-1680.

Rigoni, M., Paoli, M., Milanesi, E., Caccin, P., Rasola, A., Bernardi, P., and Montecucco, C. (2008). Snake phospholipase A2 neurotoxins enter neurons, bind specifically to mitochondria, and open their transition pores. J Biol Chem 283, 34013-34020.

Rigoni, M., Schiavo, G., Weston, A.E., Caccin, P., Allegrini, F., Pennuto, M., Valtorta, F., Montecucco, C., and Rossetto, O. (2004). Snake presynaptic neurotoxins with phospholipase A2 activity induce punctate swellings of neurites and exocytosis of synaptic vesicles. J Cell Sci 117, 3561-3570.

Sayas, C.L., Avila, J., and Wandosell, F. (2002). Regulation of neuronal cytoskeleton by lysophosphatidic acid: role of GSK-3. Biochim Biophys Acta 1582, 144-153.

Shina, R., Yates, S.L., Ghassemi, A., Rosenberg, P., and Condrea, E. (1990). Inhibitory effect of EDTA. Ca2+ on the hydrolysis of synaptosomal phospholipids by phospholipase A2 toxins and enzymes. Biochem Pharmacol 40, 2233-2239.

Spitzer, N.C., and Lamborghini, J.E. (1976). The development of the action potential mechanism of amphibian neurons isolated in culture. Proc Natl Acad Sci U S A 73, 1641-1645.

Sun, F., Anantharam, V., Zhang, D., Latchoumycandane, C., Kanthasamy, A., and Kanthasamy, A.G. (2006). Proteasome inhibitor MG-132 induces dopaminergic degeneration in cell culture and animal models. Neurotoxicology 27, 807-815.

Wang, G.X., and Poo, M.M. (2005). Requirement of TRPC channels in netrin-1-induced chemotropic turning of nerve growth cones. Nature 434, 898-904.

Westerlund, B., Nordlund, P., Uhlin, U., Eaker, D., and Eklund, H. (1992). The three-dimensional structure of notexin, a presynaptic neurotoxic phospholipase A2 at 2.0 A resolution. FEBS Lett 301, 159-164.

Yang, C.C., and Chang, L.S. (1991). Dissociation of lethal toxicity and enzymic activity of notexin from Notechis scutatus scutatus (Australian-tiger-snake) venom by modification of tyrosine residues. Biochem J 280 ( Pt 3), 739-744.

Yang, Y., Fukui, K., Koike, T., and Zheng, X. (2007). Induction of autophagy in neurite degeneration of mouse superior cervical ganglion neurons. Eur J Neurosci 26, 2979-2988.

Young, S.H., and Poo, M.M. (1983). Spontaneous release of transmitter from growth cones of embryonic neurones. Nature 305, 634-637.