20 December 2009. Researchers at Rockefeller University in New York report that they have identified a new booster of metabotropic glutamate receptor (mGluR) function in the form of a protein called Norbin. As described in Science on December 11, Norbin (also known as neurochondrin) interacts with the intracellular part of the mGluR5 type of receptor beneath the cell membrane to increase its signaling power, suggesting a possible new avenue to treating schizophrenia by modulating signaling at NMDA glutamate receptors.
In a study that goes from molecules to mice, Marc Flajolet of Paul Greengardís lab at Rockefeller led a multi-institution collaboration that characterized this protein in multiple ways, showing its influence on mGluR5 expression at the cell surface, mGluR5 signaling, synaptic plasticity, and behavior.
Researchers have been interested in promoting mGluR5 function because these receptors—through a chain of biochemical events—increase activity in NMDA receptors, which are hypothesized to be underactivated in schizophrenia (see SRF hypothesis reviews by D. Javitt and B. Moghaddam). Despite early evidence for the value of modulating NMDA function in schizophrenia (see SRF meeting report; SRF news story), the ultimate contribution of this approach remains in limbo, particularly as attempts at replication with the most promising compound—Lilly's mGluR2/3 agonist—have failed (see SRF meeting report). However, this may be due to methodologic issues, and work on NMDA receptors continues apace in academic and industry schizophrenia labs.
The new findings point to Norbin as a target that could be used to indirectly raise NMDA activity, without tinkering with the glutamate binding site for either NMDA or mGluR5 receptors.
Fishing for interactors
In looking for ways to promote mGluR5 function in the brain, first author Hong Wang of Rockefeller University and colleagues wanted to see if the brain already had something available. They used an intracellular portion of mGluR5 as bait to snag proteins liable to interact with it in the brain with a procedure known as a two-hybrid screen. They caught several proteins this way, including Norbin.
After verifying, using co-immunoprecipitation, that Norbin and mGluR5 actually interacted in the brain by catching them in the act, the researchers then found that Norbin binds mGluR5 at two intracellular sites close to the cell membrane. Mutations to these regions abolished binding with Norbin.
This coupling between the two proteins facilitated mGluR5 activity in cultured cells, as judged by the downstream products of mGluR5 activation with quisqualic acid: cells with both Norbin and mGluR5 produced 27.6 percent more inositol phosphates than those containing mGluR5 alone. Similarly, addition of Norbin to these cells led to longer-lasting calcium oscillations and more calcium peaks, both of which stem from release of calcium from intracellular stores after mGluR5 activation. The interaction between Norbin and mGluR5 was critical for this because these effects were not found in cells containing Norbin and mGluR5 that lacked the key binding site.
Norbin also boosted the amount of mGluR5 found on the surface of cultured cells, and, importantly, reducing endogenous Norbin expression in cultured neurons reduced mGluR5s at the cell surface.
Out of the petri dish
Leaving the petri dish and taking on the whole brain, the scientists showed that the pattern of Norbin expression in adult mouse brain resembled that of mGluR5, with prominent staining in the hippocampus, amygdala, septum, and nucleus accumbens. Synapses also contained both proteins, which colocalized to dendrites of hippocampal neurons.
Wang and colleagues also engineered conditional knockout (KO) mice to delete the Norbin gene postnatally in the forebrain; these mice also had lower levels of mGluR5 on the surface of neurons. Within the hippocampus, basic synaptic transmission was normal between Schaffer collaterals and CA1 neurons of these mice; however, synaptic plasticity was aberrant. In the Norbin KOs, mGluR5-dependent long-term depression was not as strong, reducing the synaptic signals to 85.4 percent of baseline, compared to the 69.9 percent found in wild-type mice. Similarly, a form of mGluR5-dependent long-term potentiation that increases synaptic signals could not be elicited from hippocampal slices taken from the Norbin KOs. These experiments suggest that the partnership with Norbin is key for mGluR5-mediated forms of synaptic plasticity.
Norbin KO mice also exhibited atypical schizophrenia-like behaviors. Though they startled to a loud noise just as much as wild-type mice, the Norbin KOs suppressed a subsequent startle response about 20 percent less in a prepulse inhibition paradigm. And in a test of the increased locomotion resulting from blocking NMDA receptors, the Norbin KOs were more sensitive, moving around about twice as much as wild-type mice. Blocking mGluR5s raised the amount of locomotion in response to the NMDA blocker in wild-type mice, but not in the Norbin KOs. This suggests that, without Norbin, mGluR5 receptors had bottomed out in their activity.
This report that Norbin, a protein that binds to mGluR5 beneath the cell surface, can substantially facilitate mGluR5 function suggests that the brain itself holds some good ideas for how to precisely modulate neurotransmission. It may be that modulating Norbin could be a strategy for treating schizophrenia, but at the very least, further research on the workings of the NMDA synapse and its many components promises to bring to light still more targets.—Michele Solis.
Wang H, Westin L, Nong Y, Birnbaum S, Bendor J, Brismar H, Nestler E, Aperia A, Flajolet M, Greengard P. Norbin is an endogenous regulator of metabotropic glutamate receptor 5 signaling. Science 326: 1554-1557. Abstract