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Synapse Formation and Function

Research Summary

Thomas Südhof is interested in how synapses are formed and function during development and in the adult. His work focuses on the role of synaptic cell-adhesion molecules in shaping synapse properties, on pre- and postsynaptic mechanisms of membrane traffic, and on impairments in synapse formation and function in neuropsychiatric disorders. To address these questions, Südhof's laboratory employs approaches ranging from biophysical and biochemical studies to the physiological and behavioral analyses of mutant mice and the in vitro derivation of human neurons.

Neurons communicate with each other by synaptic transmission at specialized intercellular junctions called synapses. Synapses form not only during development, but throughout life. Synapses transmit, process, and store information in brain. During synaptic transmission, a presynaptic neuron releases a chemical neurotransmitter that is recognized by the postsynaptic neuron. Neurotransmitter release is triggered when an action potential opens voltage-gated Ca2+ channels and Ca2+ flows into the presynaptic terminal. Ca2+ triggers neurotransmitter release by stimulating the fusion of synaptic vesicles (abundant secretory vesicles that are filled with neurotransmitters) with the presynaptic plasma membrane at the active zone (a specialized plasma membrane section that marks the synapse). Released neurotransmitters then elicit a postsynaptic signal by binding to specific receptors (Figure 1).

Work in my laboratory asks two principal sets of questions about how synapses form and function: (1) How does a nerve terminal release neurotransmitters in an exquisitely regulated manner, how is the release machinery organized at the active zone, and how is release altered during synaptic plasticity? (2) How does a presynaptic nerve terminal form a synapse on a postsynaptic neuron in the first place, how are the properties of the resulting synapses specified, and how are synapses impaired in neuropsychiatric disorders? Our overall goal is to describe the principal atomic mechanisms of these processes, i.e. to identify the molecules involved, to delineate their properties and structures, and to understand their physiological functions.

Neurotransmitter Release
To identify key molecules involved in neurotransmitter release, we set out more than two decades ago to characterize the proteins of synaptic vesicles and active zones. We then used biochemical and biophysical methods to determine the properties and atomic structures of these proteins, and employed targeted mouse mutants to examine their functions. Although many questions remain, these approaches have elucidated fundamental mechanisms of membrane fusion, neurotransmitter release, and synaptic plasticity.

Synaptic vesicle fusion is mediated by a fusion machinery containing two components: three synaptic SNARE proteins—the vesicle protein synaptobrevin/VAMP and the plasma membrane proteins SNAP-25 and syntaxin/HPC-1—and one SM protein (for Sec1/Munc18-like protein)—the soluble protein Munc18-1 (Figure 2). These components assemble into sequential complexes that are exquisitely regulated. Critical here is the role of Munc18-1—without it, no fusion occurs. The SNARE proteins are the "muscle" of the fusion reaction that provides the energy, but Munc18-1 and other SM-proteins contribute the "brawn" of the fusion reaction, effectively catalyzing fusion. This fusion mechanism, first elucidated at the synapse, appears to generally apply to all intracellular fusion reactions except for mitochondrial fusion, representing a blueprint for all membrane traffic.

At the synapse, synaptic vesicle fusion is triggered by Ca2+. Ca2+ acts by binding to the synaptic vesicle protein synaptotagmin, which contains two C2-domains that bind Ca2+ in ternary complexes with phospholipids and SNARE proteins. An emerging model suggests that before release is triggered, the Ca2+-independent interaction of synaptotagmin with SNARE complexes positions synaptotagmin for subsequent Ca2+-sensing. When Ca2+ flows into the terminal during an action potential, Ca2+-binding to the synaptotagmin C2-domains induces their association with phospholipids and opens the fusion pore to trigger release.

In these activities, synaptotagmin co-operates with a small SNARE-complex binding protein called complexin; both synaptotagmin and complexin are essential for Ca2+-triggering of release, and both act simultaneously as activators and clamps of membrane fusion to ensure precise regulation of release (Figure 2).

Different synaptotagmin genes are expressed in brain, but not all function in synaptic vesicle fusion. They perform sometimes overlapping functions (e.g., synaptotagmin-1,-2, and -9 as Ca2+-sensors for neurotransmitter release), and sometimes distinct functions even in the same neurons (e.g., synaptotagmin-10 acts as a Ca2+-sensor for IGF1 secretion in the same neurons in which synaptotagmin-1 acts as a Ca2+-sensor for neurotransmitter release). Overall, synaptotagmins are universal Ca2+-sensors for regulated exocytosis that operate throughout the body in different secretory processes.

In presynaptic terminals, neurotransmitter release occurs at the active zone, which are large juxtamembranous protein complexes whose central components are Munc13s and RIMs, proteins that we described some time ago (Figure 3). In addition, other proteins such as RIM-BPs, α-liprins, and ELKS contribute to the active zone. Munc13s and RIMs contain multiple domains that mediate the docking and priming of synaptic vesicles for exocytosis, and recruit Ca2+-channels into close proximity to the release sites via direct binding of RIMs to Ca2+- channels. Moreover, RIM and Munc13 proteins mediate use-dependent changes of release during short- and long-term synaptic plasticity, thus being crucial for learning and memory. Thus, the active zone constitutes a mosaic protein complex whose precise composition and activity state determine the properties of a synapse such as its strength and plasticity.

Synapse Formation
At a synapse, pre- and postsynaptic compartments are linked by trans-synaptic cell-adhesion molecules that in turn are coupled to the presynaptic release machinery or to postsynaptic receptors. Some time ago, we identified two trans-synaptic cell-adhesion molecules, namely neurexins and neuroligins. Neurexins are presynaptic proteins, are expressed from three genes in two principal forms, called α- and β-neurexins, and are highly polymorphic due to extensive alternative splicing (Figure 1). Neuroligins are postsynaptic proteins, are expressed from four genes, and are also alternatively spliced. The importance of neurexins and neuroligins in brain function is highlighted by the fact that mutations in their genes are repeatedly observed in patients with autism and schizophrenia—in fact, mutations in neurexin-1α may at present represent the only validated mutations predisposing to schizophrenia.

Neurexins bind to neuroligins and to other postsynaptic cell-adhesion molecules (such as LRRTMs and dystroglycan) to form heterophilic trans-synaptic junctions. This binding is tightly regulated by alternative splicing of neurexins, resulting in a trans-synaptic splice code. Experiments with "knockout" mice revealed that α-neurexins and neuroligins are not required for initial establishment of synapses, but are essential for their function. In mice lacking α-neurexins or neuroligins, synapses appear ultrastructurally normal, but synaptic transmission is severely impaired, such that the mutant mice die at birth. Deletions of individual α-neurexin and neuroligin genes showed that each isoform performs distinct but overlapping roles in synaptic transmission. For example, neuroligin-1 is essential for the normal recruitment of glutamate receptors to excitatory synapses, while neuroligin-2 is critical for the function of inhibitory synapses, and neuroligin-3 controls both excitatory and inhibitory synapses.

Although α-neurexins and neuroligins are not essential for synapse formation in vivo, overexpression of neurexins or neuroligins potently induces synapse formation in vitro, whereas acute knockdown of both neuroligins and LRRTMs suppresses synapse numbers. These effects are activity-dependent, i.e. depend on synaptic signaling, suggesting that neurexins and their ligands function in the activity-dependent organization and specification of synapses. Since in α-neurexin or neuroligin knockout mice synapses are nonfunctional, no activity-dependent changes in synapse numbers would be expected, accounting for the lack of changes in synapse numbers. The hypothesis that neurexins and neuroligins specify synapse properties is further supported by striking changes in synaptic transmission produced by point mutations in neuroligin-3. Specifically, two different missense mutations observed in neuroligin genes in patients with autism (the R451C and the R704C substitutions) dramatically altered synaptic transmission in a region-specific manner, such that the phenotype of the mutations differs between the cortex and hippocampus, and between the two mutations. Overall, these studies showed that neurexins and neuroligins are not only genuine synaptic cell-adhesion molecules that guide synapse formation by recruiting crucial components of the pre- and postsynaptic machinery to synapses, but also that their functions are activity- and context-dependent, and contribute to conferring specific properties onto synapses.

A major challenge now is to understand how neurexins shape synaptic circuits and collaborate with other trans-synaptic cell-adhesion molecules. An even more important and broader question is how synapses are formed in the first place, and what additional molecules shape their properties. Recent studies revealed that neurexins bind to many other molecules in synapse formation besides neuroligins, especially LRRTMs and cerebellins. How neural circuits are formed and how neurexins contribute to their formation are among the major questions of current neuroscience. The molecular components involved are only now beginning to be defined, with neurexins leading the way—undoubtedly many proteins will follow. Given the complexity of synaptic connectivity—with at least 1016 synapses in brain!—a combinatorial code possibly following rather simple principles may apply. Unraveling this code will provide major progress, and most certainly involve a major role of neurexins as central trans-synaptic organizers. Addressing these questions is the major current goal of my laboratory.

These studies were funded in part by grants from the National Institutes of Health and the Simons Foundation.

As of May 30, 2012

Scientist Profile

Stanford University