Core Faculty

Robert Edwards, Ph.D.

The Molecular Analysis of Neurotransmitter Release

Research Description

The regulated release of neurotransmitter by exocytosis confers many of the properties fundamental to synaptic transmission, but we still understand little about the mechanisms responsible for release.  How does exocytosis transform presynaptic electrical information into a chemical signal transmitted to the postsynaptic cell?  How does release keep up with the high rates of firing observed at many synapses?  How does it contribute to synaptic plasticity?   In many cases, the synapse faces choices dictated by the exocytotic mechanism:  synapses with a high probability of release usually show depression with repeated stimulation;  the failure to recycle transmitter after exocytosis results in greater postsynaptic signaling but depletion of the transmitter available for subsequent release.  Synapses vary in the way they cope with these limitations inherent in the exocytotic mechanism, but also take advantage of them to process information in a variety of ways.  Alterations in the release mechanism also contribute to the pathogenesis of neuropsychiatric disease.

The amount of transmitter in a secretory vesicle is considered the elementary unit in exocytotic signaling, but undergoes substantial regulation in the case of both neuromodulators such as monoamines that act by volume transmission, and the more synaptically restricted GABA and glutamate.  In general terms,  neurotransmitter packaging relies on two transport mechanisms that operate in series:  a plasma membrane reuptake system that captures released transmitter, and a vesicular transport system that refills secretory vesicles.  Previous work has described several activities responsible for vesicular neurotransmitter transport, and we have identified the three distinct families of proteins that are responsible.  We are now using these proteins as an entrypoint to understand both the recycling of transmitter and the intimately related recycling of synaptic vesicles.

The small volume of secretory vesicles imposes several surprising constraints on their filling.  In contrast to the gradients of Na+, K+ and Cl- across the plasma membrane which involve large concentrations and are generally quite stable, the transport of all classical transmitters into secretory vesicles depends on a H+ electrochemical gradient generated by an ATP-driven H+ pump that is dissipated and regenerated with each cycle of exocytosis and endocytosis.   In addition, the stoichioimetry of ionic coupling confers differences in the dependence of neurotransmitter transport on the two components of this gradient, the pH gradient and membrane potential.   These two components can be regulated independently and we are now characterizing the mechanisms responsible for their regulation in synaptic vesicles, and the effects on transmitter storage and release.   We are also developing more powerful biophysical methods to characterize the activity of vesicular neurotransmitter transporters.

We have taken advantage of the vesicular transporters to address several fundamental and long-standing questions about synaptic signaling.  Many observations have suggested that dendritic exocytosis mediates the activity-dependent retrograde release of factors such as neurotrophins from postsynaptic neurons that regulates synaptic strength.  Despite its proposed role in synaptic plasticity and homeostasis, dendritic exocytosis remains very poorly understood.  On the other hand, considerable previous work has demonstrated the regulated release of dopamine from dendrites as well as axon terminals.  Consistent with this, vesicular monoamine transporter 2 (VMAT2) localizes to dendrites as well as axons.  To understand the mechanisms of release at both sites, we have studied the trafficking of VMAT2 to synaptic vesicles and dense core vesicles in PC12 cells.  More recently, we have extended the analysis to neurons, and developed a method to image dendritic exocytosis.  We are now manipulating VMAT2 to understand the role of dendritic monoamine release in behavior.  We are also using VMAT2 as a tool  to characterize the mechanisms involved in the biogenesis of large dense core vesicles, which mediate the release of all neural peptides, peptide hormones and many growth factors.    
   In the original molecular cloning, we found that the vesicular monoamine transporter protects against the toxin MPTP, which produces a model of Parkinson’s disease (PD).  VMAT pumps the active metabolite MPP+ into secretory vesicles and thus sequesters it from its primary site of action in mitochondria.  Since dopamine is itself quite toxic, we hypothesize that VMAT also protects against the toxicity of the normal transmitter by lowering its cytoplasmic concentration.  We are thus interested in how VMAT function and by extension, cytosolic dopamine, may contribute to idiopathic PD.  Interestingly, the presynaptic protein a-synuclein has also been implicated in PD, but its role in degeneration and indeed its normal function remain unknown.  We are now studying the function of synuclein at the nerve terminal by optical imaging with several tools we have recently developed.

Synaptic vesicles at the nerve terminal belong to two functionally distinct pools, a smaller recycling pool that mediates most transmitter release under physiological conditions, and a larger reserve pool that undergoes exocytosis only with strong stimulation.  However, it is generally considered that vesicles in the two pools are identical in biochemical composition, and differ only as an accident of their history at the terminal.  Through recent work on the trafficking of vesicular glutamate transporter VGLUT1, we have uncovered two distinct recycling pathways, one that apparently corresponds to typical clathrin-mediated endocytosis, and a second pathway activated only by strong stimulation that involves the adaptor protein AP-3 and may correspond to bulk endocytosis.  We hypothesize that these two pathways produce synaptic vesicles with different biochemical composition and hence different physiological properties that may account for the behavior of recycling and reserve pool vesicles.    In addition, the results indicate the potential for the independent regulation of individual synaptic vesicle protein trafficking to the two pathways as a function of activity, a mechanism that may contribute to synaptic plasticity.   We are also using VGLUT1-pHluorin to characterize the function of synuclein in the nerve terminal.

In addition to VGLUT1 and 2, which account for glutamate release by most known glutamate neurons, we have identified a novel isoform (VGLUT3) expressed by neurons not generally considered to use glutamate as a transmitter, including serotonergic neurons in the raphe, cholinergic interneurons in the striatum, and even GABAergic interneurons in the cortex and hippocampus.  The results suggest a novel role for glutamate release by these cell populations, and we are interested in how glutamate release contributes to the neural circuits in which these cells participate.

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Current Projects

1) the biophysical properties of synaptic vesicles
2) molecular characterization of vesicular neurotransmitter transporters
3) the physiological role of somatodendritic dopamine release
4) the regulation of neurotransmitter release from dendrites
5) biogenesis of the regulated secretory pathway
6) the role of bulk endocytosis at the nerve terminal
7) the role of glutamate release from neurons that also release another transmitter
8) the function of alpha-synuclein at the nerve terminal and in Parkinson's disease

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Selected Publications

Bellocchio, E. E., Reimer, R. J., Fremeau, R. T. J., and Edwards, R. H. 2000. Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter. Science 289, 957-960.

Waites, C.L., Mehta, A., Tan, P.K., Friesen, E., Thomas, G., Edwards, R.H., Krantz, D.E. 2001. An acidic motif retains vesicular monoamine transporter 2 on large dense core vesicles. J. Cell Biol. 152, 1159-1168.

Fremeau, R.T. Jr., Troyer, M.D., Pahner, I., Nygaard, G.O., Tran, C.H., Reimer, R.J., Bellocchio, E.E., Fortin, D., Storm-Mathisen, J., Edwards, R.H. 2001. The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron 31, 247-260.

Chaudhry, F.A., Krizaj., D., Larsson, P., Reimer, R.J., Wreden, C., Storm-Mathisen, J., Copenhagen, D., Kavanaugh, M., Edwards, R.H. 2001. Coupled and uncoupled proton movement by amino acid transport system N. EMBO J. 20, 7041-7051.

Fremeau, R.T. Jr., Burman, J., Qureshi, T., Tran, C.H., Proctor, J., Johnson, J., Zhang, H., Sulzer, D., Copenhagen, D.R., Storm-Mathisen, J., Reimer, R.J., Chaudhry, F.A., Edwards, R.H. 2002. The identification of vesicular glutamate transporter 3 suggests novel modes of signalling by glutamate. Proc. Natl. Acad. Sci. USA 99, 14488-14493.

Fremeau, R.T. Jr., Kam, K., Qureshi, T., Johnson, J., Copenhagen, D.R., Storm-Mathisen, J., Chaudhry, F.A., Nicoll, R.A., Edwards, R.H. 2004. Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites. Science 304, 815-819.

Fortin, D.L., Troyer, M.D., Nakamura, K., Kubo, S.I., Anthony, M.D. and Edwards, R.H. 2004. Lipid rafts mediate the synaptic localization of a-synuclein. J. Neurosci. 24, 6715-6723.

Kubo, S.I., Nemani, V.M., Chalkley, R.J., Anthony, M.D., Hattori, N., Mizuno, Y., Edwards, R.H., Fortin, D.L 2005. A combinatorial code for the interaction of alpha -synuclein with membranes. J. Biol. Chem. 280, 31664-31672.

Fortin, D.L., Nemani, V.M., Voglmaier, S.M., Anthony, M.D., Ryan, T.A., Edwards, R.H. 2005. Neural activity controls the synaptic accumulation of a-synuclein. J. Neurosci. 25, 10913-10921.

Li, H., Waites, C.L., Staal, R.G., Dobryy, Y., Park, J., Sulzer, D.L. Edwards, R.H. 2005. Sorting of vesicular monoamine transporter 2 to the regulated secretory pathway confers the somatodendritic exocytosis of monoamines. Neuron 48, 619-633.

Voglmaier, S.M., Kam, K., Yang, H., Fortin, D.L., Hua, Z., Nicoll, R.A., Edwards, R.H. 2006. Distinct endocytic pathways control the rate and extent of synaptic vesicle recycling. Neuron 51, 71-84.

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Contact Information

Email: robert.edwards@ucsf.edu
Phone: (415) 502-5687
600 16th St.
GH-N272B
San Francisco, CA 94158-2517

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