03 Jul 2003

Synapses-those specialized junctions that facilitate transmission of action potentials between neurons-are composed of a myriad of highly sophisticated proteins. Some, like neurexins, have yet to be fully characterized. In recent issues of Nature and Nature Neuroscience, two research groups advance our understanding of these cell surface proteins, revealing essential roles played by a- and b-neurexins in synaptic transmission and assembly, respectively.

Active synapses are known to be formed when neuroligins on the surface of one cell come in contact with the axon of another. Writing in the June 8 Nature Neuroscience online, Peter Scheiffele at University of California, Berkeley, together with colleagues at Columbia University, New York, conclude that b-neurexins play a major role in this process.

Joint first authors Camin Dean and Francisco Scholl used confocal microscopy to show that neurexins and synaptic vesicles concentrate around sites of neuroligin contact. In addition, when they presented hippocampal neurons with neuroligin-coated liposomes, which are devoid of other "postsynaptic" proteins, neurexin clustered at the site of contact. This prompted the authors to ask if neurexin clustering alone was sufficient to induce synapse formation. Their answer came when they transfected hippocampal neurons with a gene that expresses neurexin-1b coupled to an extracellular tag, in this case an epitope from the vesicular stomatitis virus (VSV), and then added "multimerized" anti-VSV antibodies. The resulting clustering of b-neurexin led to a significant accumulation of synaptic vesicles while normal anti-VSV, or clustering of other cell adhesion molecules, had no such effect. The authors suggest that clusters of b-neurexin are essential for recruitment of synaptic vesicles. Such a phenomenon is not unheard of as receptor clustering plays a crucial role in the activation of T cells.

But what of the a-neurexins? There are over one thousand a-neurexin variants formed by alternative splicing of three large neurexin genes. These isoforms are known to bind a variety of both pre- and postsynaptic proteins, including the synaptic vesicle protein synaptotagmin, and postsynaptic cell adhesion molecules such as dystroglycan and the neuroligins. However, no direct evidence links a-neurexins to synaptic function. To address this, Thomas Sudhof at the Center for Basic Neuroscience, Dallas, Texas, together with colleagues at the University of Texas Southwestern Medical Center, also in Dallas, and coworkers at the Georg-August University, Gottingen, and the Ruhr-University Bochum, both in Germany, made mouse knockouts (KOs) of one, two, or all three neurexins genes. Their results are reported in the June 26 Nature.

First author Markus Missler and colleagues noticed that once born, the knockout mice had difficulty breathing. Triple KOs died within a day of delivery, double KOs only survived for a week, while single KOs, though surviving much longer, had significantly shorter life spans than did control animals (about 30 percent of single KOs died in as many days vs. only about three percent of controls). Morphologically, however, the animals looked normal and the distribution pattern and amounts of 22 different neuronal proteins were similar to control mice, indicating that loss of a-neurexins did not result in major structural changes in the central nervous systems.

So what are the neurexins doing? To answer this, Missler and colleagues examined spontaneous synaptic activity in tissue slices taken from the neocortex and brainstem-the latter being chosen because it provides the neural network essential for breathing. They found that triple KO animals had a dramatic reduction in the rate of transmissions (by over 50 percent). Double KOs had a slightly higher frequency, while single KOs produced spontaneous currents almost at the same rate as wild-type animals. Significantly, the authors found that the current amplitudes did not change, suggesting that the postsynaptic machinery was intact and that neurexins act presynaptically. In support of this idea the authors found that electrically induced synaptic transmission in the KO mice was much weaker than in wild-type mice.

Synaptic transmission occurs following an influx of calcium through voltage-gated channels. Missler used selective calcium channel blockers to examine the relationship between these ion channels and a-neurexins. When he added omega-conotoxin (a specific blocker of N-type calcium channels) to cultured brain slices from wild-type animals, the frequency of spontaneous transmission fell by almost 80 percent. However, the toxin had a progressively weaker effect on single, double, and triple knockouts, indicating that the N-type calcium channels are already malfunctioning in these animals. The authors also found that sucrose, which induces synaptic transmission independently of calcium channels, can elicit almost as good a synaptic response from triple knockout neurons as from wild-type ones.

In an accompanying News and Views, J. Troy Littleton and Morgan Sheng from MIT suggest that these results shift our view of neurexins from being part of the structural machinery to being part of the regulatory apparatus. Missler and colleagues venture that their results "reveal a link between two previously unconnected processes-synaptic cell adhesion and voltage-gated calcium signaling." Littleton and Sheng suggest that because a-neurexins can bind proteins on either side of the synapse, they can provide a means to line up calcium channels opposite postsynaptic receptors. Such an alignment would facilitate neurotransmitter release in precisely the right spot, helping the chemicals bridge the synapse and contributing to the rapidity of the transmission.-Tom Fagan.

References:
Missler M, Zhang W, Rohlmann A, Kattenstroth G, Hammer RE, Gottmann K, Sudhof TC. Alpha-neurexins couple Ca2+ channels to synaptic vesicle exocytosis. Nature. 2003 Jun 26;424(6943):939-48. Abstract

Dean C, Scholl FG, Choih J, DeMaria S, Berger J, Isacoff E, Scheiffele P. Neurexin mediates the assembly of presynaptic terminals. Nature Neurosci. 2003 Jul ;6(7):708-16. Abstract

Littleton JT, Sheng M. Neurobiology: synapses unplugged. Nature 2003 June 26;423:931-932. Abstract

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1. Missler M, Zhang W, Rohlmann A, Kattenstroth G, Hammer RE, Gottmann K, Südhof TC. Alpha-neurexins couple Ca2+ channels to synaptic vesicle exocytosis. Nature. 2003 Jun 26;423(6943):939-48. PubMed.
2. Dean C, Scholl FG, Choih J, DeMaria S, Berger J, Isacoff E, Scheiffele P. Neurexin mediates the assembly of presynaptic terminals. Nat Neurosci. 2003 Jul;6(7):708-16. PubMed.
3. Littleton JT, Sheng M. Neurobiology: synapses unplugged. Nature. 2003 Jun 26;423(6943):931-2. PubMed.