George J. Zanazzi

2nd Year Graduate Student

Department: Neurobiology & Behavior

Graduate Program: Neuroscience

Advisor: Dr. Gary Matthews

Abstract:

Title:  Mechanisms of Exocytosis at Tonic Synapses

Fast communication between neurons occurs at synapses, where vesicles in the presynaptic terminal release transmitter onto the target neuron in response to an influx of calcium. Signal propagation is particularly rapid and sustained at the first two synapses in the visual system; i.e., the output synapses of photoreceptors and bipolar cells. The presynaptic terminals of these two neurons tonically release transmitter at active zones containing ribbons, which are proteinaceous organelles that tether vesicles. While the synaptic ribbon is essential for visual function, its precise role in exocytosis is unclear. The ribbon may act like a conveyor belt that moves vesicles to the active zone. Alternatively, the ribbon may provide a scaffold for vesicles to fuse with each other in response to depolarization. While compound fusion is known to occur in several secretory cell types, definitive proof for this mechanism in synaptic vesicle exocytosis is lacking. To examine vesicle fusion mechanisms at retinal ribbon synapses, we are generating transgenic zebrafish that express sypHy, a pH-sensitive GFP attached to the lumenal end of synaptophysin, in cone photoreceptors and bipolar cells under the control of a heat shock promoter. Additional transgenic zebrafish are being generated that express sypHy under the control of the mGluR6 promoter, which we have found to drive robust, specific expression of EYFP in ON bipolar cells. In order to visualize sypHy-tagged vesicles specifically at synaptic ribbons, we are utilizing a fluorescent peptide that binds to RIBEYE, a specific component of ribbons. If homotypic vesicle fusion occurs at these synapses in response to depolarization, there should be an increase in fluorescence along the ribbon.

Membrane fusion is driven by a core SNARE complex that appears to be clamped, by a protein called complexin, in a fusion-ready state before calcium enters the presynaptic terminal and binds to synaptotagmin. Complexins 3 and 4 are enriched at ribbon-containing synapses in the mammalian retina, but their functions at these synapses are unknown. We have identified two zebrafish complexin 3 orthologs and two complexin 4 orthologs in the retina. All four orthologs are expressed in the retina by 5 days post-fertilization, coincident with the onset of visual responses in zebrafish. In particular, complexins were found to be robustly expressed in the outer and inner nuclear layers. To understand the roles of these complexins in regulating vesicle fusion, we will knockdown their expression with morpholino antisense oligos in transgenic zebrafish that express sypHy. Since complexins found at conventional synapses are thought to inhibit fast, synchronous exocytosis until they are displaced by synaptotagmin, it is possible that ribbon-associated complexins stabilize primed vesicles and clamp homotypic and heterotypic fusion before stimulation. Taken together, these studies aim to characterize cellular and molecular mechanisms of vesicle fusion at tonic synapses in the retina, and may shed light on general mechanisms of exocytosis.

Publications:
(MSTP-supported publications indicated with an *)

*Zanazzi G, Matthews G. (2007). A doubleheader in endocytosis. Neuron 56:939-942.

Koticha, D., Maurel, P., Zanazzi, G., Kane-Goldsmith, N., Basak, S., Babiarz, J., Salzer, J., Grumet, M. (2006). Neurofascin interactions play a critical role in clustering sodium channels, ankyrin G and beta IV spectrin at peripheral nodes of Ranvier. Devel. Biol. 293:1-12.

Melendez-Vasquez, C., Carey, D.J., Zanazzi, G., Reizes, O., Maurel P., and Salzer, J.L. (2005). Differential expression of proteoglycans at central and peripheral nodes of Ranvier. Glia. 52:301-308.

Taveggia C., Zanazzi, G., Petrylak, A., Yano, H., Rosenbluth, J., Einheber, S., Xu, X., Esper, R., Loeb, J., Shrager, P., Falls, D.L., Chao, M.V., Role, L., and Salzer, J.L. (2005). Type III neuregulin-1 levels determine the ensheathment fate of axons. Neuron. 47:681-694.

Gil, O.D., Zhang, L., Chen, S., Ren, Y.Q., Pimenta, A., Zanazzi, G., Hillman, D., Levitt, P. and Salzer, J.L. (2002). Complementary expression and heterophilic interactions between IgLON family members neurotrimin and LAMP. J. Neurobiol. 51:190-204.

Rambukkana, A., Zanazzi, G., Tapinos, N. and Salzer, J.L. (2002). Contact-dependent induction of demyelination by Mycobacterium leprae in the absence of immune cells. Science. 296:927- 931.

Chen, S., Gil, O.D., Ren, Y.Q., Zanazzi, G., Salzer, J.L. and Hillman, D. (2002). Neurotrimin expression during cerebellar development suggests roles in axon fasciculation and synaptogenesis. J. Neurocytol. 30:927-937.

Lustig M.*, Zanazzi G.*, Sakurai T., Blanco C., Levinson S.R., Lambert S., Grumet M., and Salzer J.L. (2001). Nr-CAM and neurofascin interactions regulate ankyrin G and sodium channel clustering at the node of Ranvier. Curr. Biol. 11:1864-1869. *Equal contribution.

Melendez-Vasquez, C., Rios, J.C., Zanazzi, G., Bretscher, A., Lambert, S. and Salzer, J.L. (2001). Nodes of Ranvier form in association with ERM-positive Schwann cell microvilli. Proc. Natl. Acad. Sci. 98:1235-1240.

Zanazzi, G., Einheber, S., Westreich, R., Hannocks, M.-J., Bedell-Hogan, D., Marchionni, M.A. and Salzer, J.L. (2001). Glial growth factor/neuregulin inhibits myelination and induces demyelination. J. Cell Biol. 152:1289-1299.

Ng, V.*, Zanazzi, G.*, Timpl, R., Talts, I.F., Salzer, J.L., Brennan, P.J. and Rambukkana, A. (2000). Role of the cell wall phenolic glycolipid-1 in the peripheral nerve predilection of Mycobacterium leprae. Cell. 103:511-524. *Equal contribution.

Tikoo, R., Zanazzi, G., Shiffman, D., Salzer, J. and Chao, M.V. (2000). Cell cycle control of Schwann cell proliferation: role of cyclin-dependent kinase-2. J. Neurosci. 20:4627-4634.

Ching, W., Zanazzi, G., Levinson, S.R. and Salzer, J.L. (1999). Clustering of neuronal sodium channels requires contact with myelinating Schwann cells. J. Neurocytol. 28:295-301.

Galbiati, F., Volonte, D., Gil, O., Zanazzi, G., Salzer, J.L., Sargiacomo, M., Scherer, P.E., Engelman, J.A., Schlegel, A., Parenti, M., Okamoto, T. and Lisanti, M.P. (1998). Expression of caveolin-1 and -2 in differentiating PC12 cells and dorsal root ganglion neurons: caveolin-2 is up-regulated in response to cell injury. Proc. Natl. Acad. Sci. 95:10257-10262.

Rambukkana, A., Yamada, H., Zanazzi, G., Mathus, T., Salzer, J.L., Yurchenco, P.D., Campbell, K.P. and Fischetti, V.A. (1998). Role of alpha-dystroglycan as a Schwann cell receptor for Mycobacterium leprae. Science. 282:2076-2079.

Gil, O.D., Zanazzi, G., Struyk, A. and Salzer, J.L. (1998). Neurotrimin mediates bifunctional effects on neurite outgrowth via homophilic and heterophilic interactions. J. Neurosci. 18:9312- 9325.

Einheber, S., Zanazzi, G., Ching, W., Scherer, S., Milner, T.A., Peles, E. and Salzer, J.L. (1997). The axonal membrane protein Caspr/neurexin IV is a component of the septate-like paranodal junctions that assemble during myelination. J. Cell Biol. 139:1495-1506.

Desser, T.S., Rubin, D.L., Muller, H.H., Qing, F., Khodor, S., Zanazzi, G., Young, S.W., Ladd, D.L., Wellons, J.A., Kellar, K.E., Toner, J.L. and Snow, R.A. (1994). Dynamics of tumor imaging with Gd-DTPA-polyethylene glycol polymers: dependence on molecular weight. J. Mag. Res. Imag. 4:467-472.

Young, S.W., Sidhu, M.K., Qing, F., Muller, H.H., Neuder, M., Zanazzi, G., Mody, T.D., Hemmi, G., Dow, W., Mutch, J.D., Sessler, J.L. and Miller R.A. (1994). Preclinical evaluation of gadolinium (III) texaphyrin complex: a new paramagnetic contrast agent for magnetic resonance imaging. Invest. Radiol. 29:330-338.

Go To Top