Scheller, Richard

Richard H. Scheller


Sudhof, Thomas

Thomas C. Südhof

Stanford University School of Medicine

For discoveries concerning the molecular machinery and regulatory mechanism that underlie the rapid release of neurotransmitters.

The 2013 Albert Lasker Basic Medical Research Award honors two scientists for their discoveries concerning rapid neurotransmitter release, a process that underlies all of the brain’s activities. Richard H. Scheller (Genentech) and Thomas C. Südhof (Stanford University School of Medicine) identified and isolated many of this reaction’s key elements, unraveled central aspects of its fundamental mechanism, and deciphered how cells govern it with extreme precision. These advances have provided a molecular framework for understanding some of the most devastating disorders that afflict humans as well as normal functions such as learning and memory.

The billions of nerve cells in our brains allow us to savor chocolate, whack a baseball, and imagine traveling at the speed of light. Their exploits tell our hearts to quicken and make us feel as if those same hearts are breaking. Their messages give us eureka moments — and let us jump out of the bathtub in response.

A biological relay system achieves these feats. Neurotransmission kicks off with an electrical pulse that runs down a nerve cell, or neuron. When that signal reaches the tip, calcium enters the cell. In response, the neuron liberates chemical messengers — neurotransmitters — which travel to the next neuron and thus pass the baton.

In the 1950s, the late Bernard Katz figured out that cells eject neurotransmitters in fixed amounts. Electron-micrographic studies by others illuminated how. Balloon-like containers — vesicles — each hold set quantities of the chemicals. Calcium incites these lipid-bound sacs to fuse with the membrane that encases the cell, and their contents spill out (see Figure).

Neurotransmission occurs astonishingly quickly — fast enough for a person to pull a hand off a hot burner or dodge an attacking mountain lion. Calcium entry can spark the release of neurotransmitter packages in less than a millisecond.

Although this general scheme had emerged by the time Südhof and Scheller began their work, its molecular details eluded scientists. No one knew what drives the vesicles to fuse with the cell membrane or how calcium provokes that event.

Actors at the membrane

In the late 1980s, Scheller and Südhof set out independently to unveil the molecular underpinnings of the process. Reasoning that nerve terminals would hold the reaction’s components, the scientists decided to purify and characterize proteins at that site. Focusing first on vesicles, Scheller, then at Stanford University School of Medicine, discovered what would turn out to be an essential piece of the fusion apparatus: vesicle-associated membrane protein (VAMP), which he obtained from the electric organ of a marine ray. The following year, Südhof, then at the University of Texas Southwestern Medical Center in Dallas, isolated the same protein from rat brain and called it synaptobrevin.

Südhof next purified and studied another vesicle protein, synaptotagmin. When calcium is present, this protein binds to phospholipids, major constituents of membranes. Scheller established that synaptotagmin clutches a brain protein that he named syntaxin. In contrast to vesicle-dwelling synaptotagmin and VAMP/synaptobrevin, syntaxin concentrates in the cell membrane — at spots where neurotransmitters are released from nerve cells.

Independently, James Rothman (Yale University) (Lasker Basic Medical Research Award, 2002) had been exploring how substances are ferried from one place to another inside cells. In that process, too, transport vesicles deliver their contents by merging their membranes with those of the target compartment. Rothman had proposed that one of the proteins necessary for his experimental system and for fusion in live yeast cells, NSF, attaches to the membrane through a second protein and its as-yet-unidentified collaborators. He set out to unearth these hypothetical proteins, which he called SNAREs.

To nab them, Rothman sought proteins in rat brains that adhere to NSF through its partner. Three proteins stuck: VAMP/synaptobrevin, syntaxin, and SNAP-25, a protein reported previously to reside on cell membranes of nerve termini.

Two separate lines of inquiry thus pointed at the same three molecules. The results suggested that they promote neurotransmitter release by fostering fusion, but provided only indirect evidence. Meanwhile, a clear link to their physiological function had materialized from a different direction. Scientists had long known that certain bacterial toxins thwart neurotransmission, and in 1992, Cesare Montecucco (University of Padua) showed that botulinum and tetanus toxins block neurotransmitter release by clipping off a chunk of VAMP/synaptobrevin. Subsequent work by Montecucco, Südhof, and Reinhard Jahn (Max Planck Institute, G ttingen) demonstrated that additional neurotoxins attack syntaxin and SNAP-25.

These observations established that the three proteins are vital for neurotransmission. The mechanism of fusion was murky, however, as was the way in which calcium triggers the process.

Findings fuse

The previous work had shown that VAMP/synaptobrevin, syntaxin, and SNAP-25 bind to the NSF conglomeration, but not whether they do so alone or together. In 1993, Scheller, in collaboration with Rothman, found that VAMP/synaptobrevin, syntaxin, and SNAP-25 associate stably with one another to form the so-called ‘SNARE complex’. The team also showed that NSF rips apart this assemblage. Scientists later realized that this phenomenon helps recycle the molecular machinery.

Neurotransmitter illustration

In the meantime, researchers were defining the precise interactions among these proteins and discerning how the associations might instigate fusion (see Figure for current model). Scheller found, for instance, that VAMP/synaptobrevin on vesicles clasps plasma membrane-bound syntaxin, and that SNAP-25 helps them pair up. Work by Scheller, Jahn, John Heuser (Washington University School of Medicine, St. Louis), and others advanced the concept that VAMP/synaptobrevin and syntaxin form coils that wrap around each other along their lengths, thus drawing together the membranes in which they’re embedded. Axel Brunger (Yale University) and Jahn confirmed and extended these ideas when they reported the high-resolution structure of VAMP/synaptobrevin, syntaxin, and SNAP-25 in 1998. The results supported a scenario in which the proteins zipper together, eventually forcing fusion (see Figure).

Control mechanisms calcify

These and other observations were fleshing out the basic mechanism of fusion, but they also highlighted crucial open questions. Uncontrolled, the reaction would result in rampant and constant neurotransmitter release. Although scientists knew that the system does not launch until calcium arrives, the details remained obscure. It was clear, however, that the extremely short time between calcium influx and neurotransmitter discharge would not permit assembly of a multi-protein machine. The device must lie on the brink of its fusion-competent state, waiting for calcium to push it over the edge, presumably by a protein that senses this ion.

Südhof’s original observations on synaptotagmin tantalized him because they indicated that the protein performs calcium-dependent activities. Perhaps, he speculated, calcium prompts it to facilitate fusion. In 1993, he showed that two regions of synaptotagmin bind calcium, and this property allows it to efficiently grasp phospholipids. These results bolstered the protein’s candidacy as the calcium sensor.

In 1994, he generated mice that lack operational synaptotagmin. Although the animals died soon after birth, neurons from their embryos could be studied. Südhof found that their core fusion machinery remained functional, but unable to respond to calcium.

In an elegant set of experiments, Südhof ruled out the possibility that loss of synaptotagmin impairs calcium entry into the cell or hinders some other event peripheral to calcium perception. He generated a series of mice, each of which carried a synaptotagmin with altered calcium-binding affinity. Synaptotagmin’s affinity for the ion correlated with the calcium sensitivity of neurotransmission. Increasing calcium avidity, for instance, decreased by approximately the same amount how much calcium was needed for neurotransmitter release. These results confirmed the notion that synaptotagmin functions as the calcium sensor.

Meanwhile, another layer of regulation was surfacing. Südhof had discovered a protein, complexin, that strongly adheres to the VAMP/synaptobrevin, syntaxin, and SNAP-25 bundle. Subsequent work established that it plays an essential role in calcium-regulated neurotransmitter release. Complexin holds the partially zippered SNARE complex in a form that is poised to trigger fusion, yet inactive until the crucial next step: Calcium binds to synaptotagmin and spurs it to displace complexin, which instantly drives the reaction (see Figure).

Medical implications

Communication within the brain influences how we think and who we are. Defects in the process contribute to schizophrenia, depression, bipolar disorder, and many other pathological conditions. Studies of these illnesses have not yet indicted misbehaving components of the fusion complex itself, but scientists are beginning to uncover connections between this equipment and serious diseases. For example, α-synuclein, a protein that has been linked to Parkinson disease and other neurodegenerative disorders, helps assemble the fusion apparatus and protects animals from age-related neurological problems. Flaws in Munc18, another protein that Südhof and Scheller implicated in neurotransmitter release and that is indispensable for it, have been associated with Ohtahara syndrome, a severe epileptic disorder that strikes during infancy and causes seizures and mental retardation.

By systematically exposing and analyzing the proteins involved in neurotransmitter release, Südhof and Scheller have transformed our description of the process from a rough outline to a series of nuanced molecular transactions. Their work has revealed the elaborate orchestrations that lie at the crux of our most simple and sophisticated neurobiological activities.

by Evelyn Strauss

Key publications of Richard H. Scheller

Trimble, W.S., Cowan, D.M., and Scheller, R.H. (1988). VAMP-1: A synaptic vesicle-associated integral membrane protein. Proc. Natl. Acad. Sci. USA. 85, 4538-4542.

Bennett, M.K., Calakos, N., and Scheller, R.H. (1992). Syntaxin: A synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science. 257, 255-259.

Söllner, T., Bennett, M.K., Whiteheart, S.W., Scheller, R.H., and Rothman, J.E. (1993). A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell. 75, 409–418.

Calakos, N., Bennett, M.K., Peterson, K.E., and Scheller, R.H. (1994). Protein-protein interactions contributing to the specificity of intracellular vesicular trafficking. Science. 263, 1146-1149.

Lin, R.C. and Scheller, R.H. (1997). Structural organization of the synaptic exocytosis core complex. Neuron. 19, 1087-1094.

Chen, Y.A., Scales, S.J., Patel, S.M., Doung, Y.-C., and Scheller, R.H. (1999). SNARE complex formation is triggered by Ca2+ and drives membrane fusion. Cell. 97, 165-174.

Key publications of Thomas C. Südhof

Perin, M.S., Fried, V.A., Mignery, G.A., Jahn, R., and Südhof, T.C. (1990). Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature. 345, 260-263.

Brose, N., Petrenko, A.G., Südhof, T.C., and Jahn, R. (1992). Synaptotagmin: A calcium sensor on the synaptic vesicle surface. Science. 256, 1021-1025.

McMahon, H.T., Missler, M., Li, C., and Südhof, T.C. (1995). Complexins: Cytosolic proteins that regulate SNAP receptor function. Cell. 83, 111-119.

Fernández-Chacón, R., Königstorfer, A., Gerber, S.H., Garcia, J., Matos, M.F., Stevens, C.F., Brose, N., Rizo, J. Rosenmund, C., and Südhof, T.C. (2001). Synaptotagmin I functions as a calcium regulator of release probability. Nature. 410, 41-49.

Rhee, J.-S., Li, L., Shin, O.-H., Rah, J.-C., Rizo, J., Südhof, T.C., and Rosenmund, C. (2005). Augmenting neurotransmitter release by enhancing the apparent Ca2+ affinity of synaptotagmin 1. Proc. Natl. Acad. Sci. USA. 102, 18664–18669.

Tang, J., Maximov, A., Shin, O.-H., Dai, H., Rizo, J., and Südhof, T.C. (2006). A complexin/synaptotagmin-1 switch controls fast synaptic vesicle exocytosis. Cell. 126, 1175-1187.

Review articles by Scheller and Südhof on neurotransmitter release

Bennett, M.K. and Scheller, R.H. (1993). The molecular machinery for secretion is conserved from yeast to neurons. Proc. Natl. Acad. Sci. USA. 90: 2559-2563.

Südhof, T.C. (1995). The synaptic vesicle cycle: A cascade of protein-protein interactions. Nature. 375, 645-653.

Lin, R.C. and Scheller, R.H. (2000). Mechanisms of synaptic vesicle exocytosis. Annu. Rev. Cell Dev. Biol. 16, 19-49.

Südhof, T.C. (2012). Calcium control of neurotransmitter release. Cold Spring Harb. Perspect. Biol. 4:a011353, 1-15.

Award presentation by Eric Kandel

Award presentation by Eric KandelWe are who we are because of our brain and its ability to acquire and store new information. This ability derives from the remarkable capabilities of the 100 billion signaling units in the brain called nerve cells — or neurons — and in particular from the ability of these nerve cells to communicate with one another through specialized contact points called synapses. A synapse has three components: a pre-synaptic terminal contributed by the sending neuron, a post-synaptic receptive component contributed by the receiving cell and a synaptic cleft — the space between the pre- and post-synaptic components. Synapses operate by means of a chemical called a neurotransmitter that is released from the presynaptic terminal by a process involving the influx of Ca2+, whose molecular bases our two Lasker Basic Science Prize winners, Richard Scheller and Thomas Sudhof, have pioneered in defining.

Because of its critical importance for understanding the brain, its role in our capability to learn and to remember, and the many neurological and psychiatric disorders that involve synapses, a molecular understanding of chemical synaptic transmission has been one of the holy grails of neuroscience.

The initial steps in addressing this question in the modern era were taken in 1951 when Bernard Katz, demonstrated that a chemical transmitter is released from the end of the neuron from a structure called the presynaptic terminal. It is sent not as a single molecule but as a multi-molecular packet, which Katz called a quantum. We now know that each quantum, consisting of a collection of around 5000 transmitter molecules, is contained in a little round organelle in the presynaptic terminal that Sanford Palay and George Palade had earlier discovered and called the “synaptic vesicl.e” Neurotransmitter is released from these synaptic vesicles to the outside of the neuron in response to the influx of Ca2+ into the presynaptic terminal. This influx occurs when Ca2+ channels open in response to an electrical signal, called the action potential that is conducted to the sending neuron’s presynaptic terminal. For this work Bernard Katz was awarded the Nobel Prize in 1970.

The next major advance which moved this analysis from a cell physiological to a molecular level was accomplished by Scheller and Südhof who made overlapping contributions that characterized the proteins that controlled the two key steps of transmitter release: 1) They showed the mechanism by which the vesicle is mobilized to the release sites of the presynaptic terminal, where the synaptic vesicle first fuses with the membrane of the sending neuron and then leaves the cell, and 2) they also discovered how Ca2+ drives the vesicle to release its contents. That these two scientists have accomplished so much is all the more remarkable given the painfully difficult circumstances in which they had to work in the early phases of their careers.

Let me begin with Richard Scheller. Richard had the catastrophic misfortune of beginning his scientific career in neuroscience in the years of 1981 and 1982 by working with Richard Axel and myself. Need I say more? Given how little one of those two knew about the brain and how little the other new about molecules, it is amazing that Scheller survived, much less prospered in science.

Beginning in 1988 Scheller, then at Stanford, succeeded in characterizing several key proteins necessary for synaptic vesicle fusion with the presynaptic membrane, the prerequisite step for neurotransmitter release. In 1988, Scheller identified the first key vesicle membrane protein, which he termed VAMP, now also known as synaptobrevin. Then in 1992 he cloned syntaxin, one of what proved to be two active zone plasma membrane proteins important for neurotransmitter release. Because the two molecules bound to one another Scheller proposed that VAMP, the synaptic vesicle protein, bridges to syntaxin, the plasma membrane protein — thereby providing a scaffold onto which the molecular machinery that catalyzes membrane fusion can be assembled.

In an important experiment published one year later, Jim Rothman, a Lasker Prize winner in 2002 honored for his work on fusion in non-neuronal cells, provided the experimental support for Scheller’s idea by demonstrating that Scheller’s VAMP, or as Rothman called it the v-SNARE, interacted with Scheller’s syntaxin or t-SNARE, together with a second t-SNARE, to initiate fusion.

Scheller and Rothman then collaborated and found that the three SNAREs form a stable complex, which can be disassembled when ATP is hydrolyzed by another protein in the system. In 1999, Scheller demonstrated that calcium triggering and the full formation of the SNARE complex are closely associated, thereby providing the first direct evidence that SNARE complex formation drives the fusion event responsible for neurotransmitter release and the consequent communication between neurons.

Now let me turn to Südhof. His life also was no bed of roses. Südhof had the misfortune of working as a postdoctoral fellow from 1982 to 1984 with Brown and Goldstein. If you know them at all — I also need not say more.

Südhof added new insights to the mechanisms of rapid neurotransmitter release by bringing to the problem two new approaches. First, he combined membrane protein biochemistry with molecular biological techniques to identify synaptic vesicle and other key proteins, several in collaboration with Reinhard Jahn. Second, Südhof pioneered the use of genetically modified mice to examine the functional role of these skey proteins and the machinery that regulates the calcium mechanism of neurotransmitter release.

In the first approach, in 1990 Südhof and Jahn cloned a vesicle-associated protein called synaptotagmin-l and hypothesized that synaptotagmin was the Ca2+ sensor for synaptic vesicle fusion. Südhof next carried out an elegant series of biochemical, biophysical, physiological and genetic studies to conclusively prove this hypothesis.

In the second approach, Südhof created a large number of genetically modified mice which lacked synaptotagmin. He found that these mice had selective loss of the fast Ca2+-triggered synaptic vesicle release. These and other studies provided compelling genetic evidence that Ca2+ binding to synaptotagmin drives fast Ca2+-triggered transmitter release.

Südhof next addressed the question of how Ca2+ binding to synaptotagmin promotes release. Based on his biochemical studies, Südhof developed a model whereby the SNARE complex forms at the synapse prior to actual Ca2+-triggered release. He proposed that upon Ca2+ binding, synaptotagmin opens a fusion pore at the site of the SNARE complex formation. This model was again supported by studies of a mouse model.

At a synapse, neurotransmitter release requires localization of Ca2+ channels to presynaptic active zones. How Ca2+ channels are recruited to active zones where vesicles bind and are released remained unknown. Recent studies by Südhof describe a scaffolding molecule called RIM that helps localize Ca2+ channels to the presynaptic active zone, thereby enabling fast triggering of neurotransmitter release at a synapse.

Together Südhof and Scheller have given us a new level of understanding of how synapses — the key components of our mental function — release chemical transmitter from their presynaptic terminals in response to a calcium signal. It is therefore with pleasure and pride that the Lasker Jury recognize Tom and to Richard, and that the Lasker Foundation awards them the Albert Lasker Basic Science Award for the year 2013.

Acceptance remarks

Interview with Richard H. Scheller and Thomas C. Südhof

Video Credit: Susan Hadary