17 May 2010. Known for partnering with other proteins to shape synapses, neurexins may powerfully regulate synapses all by themselves, according to a report in the May 13 issue of Neuron. Led by Thomas Südhof at Stanford University, the researchers found that neurexins in vitro—without help from their usual partners—suppress signals flowing through GABAA receptors, which are responsible for the bulk of inhibitory synaptic transmission in the brain, and that neurexins can physically interact with subunits of the GABAA receptor.
These findings bring together two previously unrelated areas in schizophrenia research—the recently identified candidate susceptibility gene for neurexin (Walsh et al., 2008) and the long-standing interest in GABA dysfunction in the disorder (see SRF related news story and SRF interview with David Lewis). GABAA receptors have, themselves, been linked to the disorder via several positive genetic association studies (see SZGene entry for GABAA). The new results also support the idea that meddling with cell adhesion molecules like neurexin can upset the balance of excitatory and inhibitory synaptic transmission in the brain. Previous studies have suggested such an imbalance for schizophrenia and autism (Etherton et al., 2009; Tabuchi et al., 2007).
Neurexins reside primarily on the pre-synaptic side of a synapse, and their extracellular portions stick out into the space between neurons. There they can form trans-synaptic bridges with post-synaptic cell adhesion molecules like neuroligins or LRRTM2 (see SRF related news story) to influence synapse assembly and function (Südhof, 2008). But the new results, obtained in reduced and tightly controlled experimental preparations, lead the authors to propose that pre-synaptically located neurexins can bypass these partnerships, reaching across to bind GABAA receptors on the post-synaptic side and impeding the development of their inhibitory signals. This newfound ability reinforces the emerging picture of neurexins as multifunctional proteins that influence synapse maturation and function.
First author Chen Zhang and colleagues stumbled upon neurexin's suppressive ability when testing whether boosting levels of neurexin in cultured neurons would increase synapse numbers. Synapse numbers did not change, but the synapses themselves were different: neurexin overexpression halved the size of currents flowing through GABAA receptors compared to controls. The suppression was specific to inhibitory synapses, too, leaving currents in excitatory synapses unchanged. All neurexin types tested—1α, 1β, 2β, and 3β—induced a similar suppression.
To address whether neurexins blocked the maturation of new synapses or disrupted the fully developed ones, the researchers studied the time course of neurexin's effect in neurons maintained in vitro. While control neurons progressively increased inhibitory current size over four days, those transfected with neurexin did not, holding steady at their initial size. This indicated that neurexin did not impair existing synapses, but instead prevented the maturation process that increases synaptic strength in newly formed synapses.
The researchers then tested whether neurexin's other binding partners played a role in this suppression, particularly neuroligin-2, which localizes to inhibitory synapses. Multiple approaches indicated that neurexin was acting alone: neurons taken from knockout mice missing neuroligin-2 still showed the neurexin-induced suppression, and a neurexin mutant that cannot bind neuroligins could still induce the suppression. Other experiments ruled out involvement of LRRTM2 or dystroglycan.
Go directly to GABAA receptors
Several experiments narrowed in on where neurexin exerts its effect, and found it acts at the surface of neurons. Notably, adding neurexin to the cell culture medium was also a potent way to suppress GABAergic responses. Similarly, a neurexin mutant lacking the cytoplasmic tail of the protein could induce the suppression, whereas a neurexin mutant lacking the extracellular portion could not.
If neurexin is acting alone, with its extracellular portion on the extracellular side of the synapse, might it be directly interacting with the components of inhibitory synapses? Researchers tested this with affinity chromatography experiments in which neurexin molecules were used to fish for binding partners in brain extracts. Neurexin bound substantial amounts of the α1 subunit of GABAA receptors, and other experiments found that the interaction involved the extracellular portions of both proteins, and that it was tight and specific with micromolar affinity, consistent with direct binding between them.
To see if this interaction was sufficient to decrease GABAergic responses, the researchers turned to a stripped-down preparation consisting only of non-neuronal cells, GABAA receptors, and neurexin. The cells were made to express GABAA receptors on their surface, and when they were transfected with neurexin, they showed smaller responses to GABA than did control cells without neurexin. Similarly, incubating the non-neuronal cells with purified neurexin for 48 hours was also sufficient to induce the suppression.
This go-it-alone action adds yet another potential function for neurexin at the synapse in vivo. Combined with the thousands of neurexin splice variants, the number of different types of neurexin interaction could be vast. Each interaction may dictate specific synapse types in specific cell types in the brain, a possibility that could help shed light on how seemingly brainwide synaptic defects may translate into the circuit-specific anomalies relevant to schizophrenia and other brain disorders. Accordingly, the authors point out that future experiments will have to tease these apart by finding neurexin mutations that selectively impair one type of interaction without interfering with the others.—Michele Solis.
Zhang C, Atasoy D, Araç D, Yang X, Fucillo MV, Robison AJ, Ko J, Brunger AT, Südhof TC. Neurexins physically and functionally interact with GABAA receptors. Neuron. 2010 May 13; 66: 403-416.