Abstract
Clathrin assembly proteins AP180 and CALM regulate the assembly of clathrin-coated vesicles (CCVs), which mediate diverse intracellular trafficking processes, including synaptic vesicle (SV) recycling at the synapse. Although studies using several invertebrate model systems have indicated a role for AP180 in SV recycling, less is known about AP180’s or CALM’s function in the synapse of mammalian neurons. In this study, we examined synapses of rat hippocampal neurons in which the level of AP180 or CALM had been reduced by RNA interference (RNAi). Using light microscopy, we visualized synaptic puncta in these AP180- or CALM-reduced neurons by co-expressing Synaptophysin::EGFP (Syp::EGFP). We found that neurons with reduced AP180 or reduced CALM had smaller Syp::EGFP-illuminated puncta. Using electron microscopy, we further examined the ultrastructure of the AP180- or CALM-reduced presynaptic terminals. We found that SVs became variably enlarged in both the AP180-reduced and CALM-reduced presynaptic terminals. Lower AP180 and CALM also reduced the density of SVs and the size of SV clusters. Our findings demonstrate that in the presynaptic terminals of hippocampal neurons, AP180 and CALM have a similar role in regulating synaptic vesicles. This overlapping activity may be necessary for high-precision and high-efficacy SV formation during endocytosis.
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References
Ahle, S., & Ungewickell, E. (1986). Purification and properties of a new clathrin assembly protein. EMBO Journal, 5, 3143–3149.
Andreae, L. C., Fredj, N. B., & Burrone, J. (2012). Independent vesicle pools underlie different modes of release during neuronal development. Journal of Neuroscience, 32, 1867–1874.
Banker, G., & Cowan, W. M. (1977). Rat hippocampal neurons in dispersed cell culture. Brain Research, 126, 397–425.
Brett, T. J., & Traub, L. M. (2006). Molecular structures of coat and coat-associated proteins: Function follows form. Current Opinion in Cell Biology, 18, 395–406.
Brodsky, F. M., Chen, C. Y., Knuehl, C., Towler, M. C., & Wakeham, D. E. (2001). Biological basket weaving: Formation and function of clathrin-coated vesicles. Annual Reviews of Cell and Developmental Biology, 17, 517–568.
Bushlin, I., Petralia, R. S., Wu, F., Harel, A., Mughal, M. R., Mattson, M. P., et al. (2008). Clathrin assembly protein AP180 and CALM differentially control axogenesis and dendrite outgrowth in embryonic hippocampal neurons. Journal of Neuroscience, 28, 10257–10271.
Dittman, J., & Ryan, T. A. (2009). Molecular circuitry of endocytosis at nerve terminals. Annual Reviews of Cell and Developmental Biology, 25, 133–160.
Dreyling, M. H., Martinez-Climent, J. A., Zheng, M., Mao, J., Rowley, J. D., & Bohlander, S. K. (1996). The t(10;11)(p13;q14) in the U937 cell line results in the fusion of the AF10 gene and CALM, encoding a new member of the AP-3 clathrin assembly protein family. Proceedings of the National Academy of Sciences, 93, 4804–4809.
Ford, M. G., Pearse, B. M., Higgins, M. K., Vallis, Y., Owen, D. J., Gibson, A., et al. (2001). Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. Science, 291, 1051–1055.
Harel, A., Wu, F., Mattson, M. P., Morris, C. M., & Yao, P. J. (2008). Evidence for CALM in directing VAMP2 trafficking. Traffic, 9, 417–429.
Kaech, S., & Banker, G. (2006). Culturing hippocampal neurons. Nature Protocols, 1, 2406–2415.
Kay, A. R., Alfonso, A., Alford, S., Cline, H. T., Holgado, A. M., Sakmann, B., et al. (1999). Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat. Neuron, 24, 809–817.
Koo, S. J., Markovic, S., Puchkov, D., Mahrenholz, C. C., Beceren-Braun, F., Maritzen, T., et al. (2011). SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia (CALM) and AP180 at synapses. Proceedings of the National Academy of Sciences, 108, 13540–13545.
Lafer, E. M. (2002). Clathrin-protein interactions. Traffic, 3, 513–520.
Li, J., Wang, Y., Chiu, S. L., & Cline, H. T. (2010). Membrane targeted horseradish peroxidase as a marker for correlative fluorescence and electron microscopy studies. Front Neural Circuits, 26, 4–6.
Mao, Y., Chen, J., Maynard, J. A., Zhang, B., & Quiocho, F. A. (2001). A novel all helix fold of the AP180 amino-terminal domain for phosphoinositide binding and clathrin assembly in synaptic vesicle endocytosis. Cell, 104, 433–440.
Mattson, M. P., Dou, P., & Kater, S. B. (1988). Outgrowth-regulating actions of glutamate in isolated hippocampal pyramidal neurons. Journal of Neuroscience, 8, 2087–2100.
McMahon, H. T., & Boucrot, E. (2011). Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nature Reviews Molecular Cell Biololgy, 12, 517–533.
Meyerholz, A., Hinrichsen, L., Groos, S., Esk, P. C., Brandes, G., & Ungewickell, E. J. (2005). Effect of clathrin assembly lymphoid myeloid leukemia protein depletion on clathrin coat formation. Traffic, 6, 1225–1234.
Mitchell, N., Petralia, R. S, Yao, P. J., Currier, D. G., Wang, Y. X., Kim, A., et al. (2012). Sonic hedgehog regulates presynaptic terminal size, ultrastructure and function in hippocampal neurons. Journal of Cell Science (Epub ahead of print).
Morgan, J. R., Zhao, X., Womack, M., Prasad, K., Augustine, G. J., & Lafer, E. M. (1999). A role for the clathrin assembly domain of AP180 in synaptic vesicle endocytosis. Journal of Neuroscience, 19, 10201–10212.
Morgan, J. R., Augustine, G. J., & Lafer, E. M. (2002). Synaptic vesicle endocytosis: the races, places, and molecular faces. NeuroMolecular Medicine, 2, 101–114.
Morris, S. A., Schroder, S., Plessmann, U., Weber, K., & Ungewickell, E. (1993). Clathrin assembly protein AP180: Primary structure, domain organization and identification of a clathrin binding site. EMBO Journal, 12, 667–675.
Nonet, M. L., Holgado, A. M., Brewer, F., Serpe, C. J., Norbeck, B. A., Holleran, J., et al. (1999). UNC-11, a Caenorhabditis elegans AP180 homologue, regulates the size and protein composition of synaptic vesicles. Molecular Biology of the Cell, 10, 2343–2360.
Petralia, R. S., & Wenthold, R. J. (1999). Immunocytochemistry of NMDA receptors. Methods Molecular Biololgy, 128, 73–92.
Petralia, R. S., & Yao, P. J. (2007). AP180 and CALM in the developing hippocampus: Expression at the nascent synapse and localization to trafficking organelles. Journal of Comparative Neurology, 504, 314–327.
Petralia, R. S., Wang, Y. X., Hua, F., Yi, Z., Zhou, A., Ge, L., et al. (2010). Organization of NMDA receptors at extrasynaptic locations. Neuroscience, 167, 68–87.
Robinson, M. S. (2004). Adaptable adaptors for coated vesicles. Trends in Cell Biology, 14, 167–174.
Royle, S. J., & Lagnado, L. (2010). Clathrin-mediated endocytosis at the synaptic terminal: Bridging the gap between physiology and molecules. Traffic, 11, 1489–1497.
Schmid, E. M., & McMahon, H. T. (2007). Integrating molecular and network biology to decode endocytosis. Nature, 448, 883–888.
Schwartz, C. M., Cheng, A., Mughal, M. R., Mattson, M. P., & Yao, P. J. (2010). Clathrin assembly proteins AP180 and CALM in the embryonic rat brain. Journal of Comparative Neurology, 518, 3803–3818.
Staras, K., Branco, T., Burden, J. J., Pozo, K., Darcy, K., Marra, V., et al. (2010). A vesicle superpool spans multiple presynaptic terminals in hippocampal neurons. Neuron, 66, 37–44.
Tebar, F., Bohlander, S. K., & Sorkin, A. (1999). Clathrin assembly lymphoid myeloid leukemia (CALM) protein: Localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated traffic. Molecular Biology of the Cell, 10, 2687–2702.
Ungewickell, E. J., & Hinrichsen, L. (2007). Endocytosis: Clathrin-mediated membrane budding. Current Opinion in Cell Biology, 19, 417–425.
Williams, M. E., Wilke, S. A., Daggett, A., Davis, E., Otto, S., Ravi, D., et al. (2011). Cadherin-9 regulates synapse-specific differentiation in the developing hippocampus. Neuron, 71, 640–655.
Wu, F., Matsuoka, Y., Mattson, M. P., & Yao, P. J. (2009). The clathrin assembly protein AP180 regulates the generation of amyloid-beta peptide. Biochemical and Biophysical Research Communications, 385, 247–250.
Yao, P. J., Zhang, P., Mattson, M. P., & Furukawa, K. (2003). Heterogeneity of endocytic proteins: Distribution of clathrin adaptor proteins in neurons and glia. Neuroscience, 121, 25–37.
Yao, P. J., Petralia, R. S., Bushlin, I., Wang, Y., & Furukawa, K. (2005). Synaptic distribution of the endocytic accessory proteins AP180 and CALM. Journal of Comparative Neurology, 481, 58–69.
Ye, W., & Lafer, E. M. (1995). Bacterially expressed F1–20/AP-3 assembles clathrin into cages with a narrow size distribution: Implications for the regulation of quantal size during neurotransmission. Journal of Neuroscience, 41, 15–26.
Zhang, B., Koh, Y. H., Beckstead, R. B., Budnik, V., Ganetzky, B., & Bellen, H. J. (1998). Synaptic vesicle size and number are regulated by a clathrin adaptor protein required for endocytosis. Neuron, 21, 1465–1475.
Zhou, S., Tannery, N. H., Yang, J., Puszkin, S., & Lafer, E. M. (1993). The synapse- specific phosphoprotein F1–20 is identical to the clathrin assembly protein AP-3. Journal of Biological Chemistry, 268, 12655–12662.
Acknowledgments
We thank the anonymous reviewers for helpful suggestions. We also thank Dr. Jane Sullivan for Synaptophysin::EGFP and Dr. Hollis T. Cline for the EGFP::mHRP construct. This work was supported by the Intramural Research Programs of the NIA/NIH and NIDCD/NIH.
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Petralia, R.S., Wang, YX., Indig, F.E. et al. Reduction of AP180 and CALM Produces Defects in Synaptic Vesicle Size and Density. Neuromol Med 15, 49–60 (2013). https://doi.org/10.1007/s12017-012-8194-x
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DOI: https://doi.org/10.1007/s12017-012-8194-x