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Meet Norbin—Behind the Scenes Player at mGluR5

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 Greengard’s 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.

Reference:
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

Comments on Related News


Related News: ICOSR 2007—Glutamate Regulator May Be Alternative to D2 Blockers

Comment by:  Patricia Estani
Submitted 21 May 2007
Posted 21 May 2007

In the field of the psychopharmacology of schizophrenia, a lot of research work has been done on dopaminergic systems. Thus, this research news is excellent news because it explores an alternative neurotransmission system in schizophrenia, the glutamatergic system. Since the work of Dr. Bita Moghaddam in 1998, published in Science, a lot of research studies have turned to the important role of glutamate in schizophrenia. More studies are needed to focus on the exact role of this neurotransmitter.

View all comments by Patricia Estani

Related News: ICOSR 2007—Glutamate Regulator May Be Alternative to D2 Blockers

Comment by:  Joseph Neale
Submitted 14 July 2007
Posted 14 July 2007

The pioneering research over the past decade on group II metabotropic glutamate receptor (mGluR) agonists from the Lilly Labs and Bita Moghaddam's research group has provided a strong foundation for the view that activation of these receptors reduces schizophrenia-like behaviors in the PCP and amphetamine models. These phase 2 clinical trials bring mGluR agonists one step closer to clinical use as therapy or co-therapy.

These same data provide the foundation for current and future research aimed at increasing the concentration of the peptide transmitter, N-acetylaspartylglutamate (NAAG) in the synaptic cleft by systemic administration of NAAG peptidase inhibitors. NAAG is the third most prevalent transmitter in the mammalian nervous system and a selective group II mGluR agonist with preference for mGluR3 (Neale et al., 2005). Our research group demonstrated that a NAAG peptidase inhibitor substantially reduces positive and negative behaviors induced in PCP models of schizophrenia (Olszewski et al., 2007; Olszewski et al., 2004). These NAAG peptidase inhibition studies parallel the preclinical studies from Lilly and Bita Moghaddam on mGluR agonists in animal models of schizophrenia. Since NAAG is an endogenous transmitter, it can be argued that elevating its levels following synaptic release by reducing the rate of its inactivation (analogous to SSRI and serotonin) is a more physiologic means of activating the mGluR, and thus this may be better tolerated with fewer side effects than the continuous receptor activation that is obtained via systemic administration of a receptor agonist.

These phase 2 clinical trials clearly brighten the prospects for both lines of new drug development for treatment of schizophrenia.

References:

Neale JH, Olszewski RT, Gehl LM, Wroblewska B, Bzdega T. The neurotransmitter N-acetylaspartylglutamate in models of pain, ALS, diabetic neuropathy, CNS injury and schizophrenia. Trends Pharmacol Sci. 2005 Sep;26(9):477-84. Review. Abstract

Olszewski RT, Wegorzewska MM, Monteiro AC, Krolikowski K, Zhou J, Kozikowski AP, Long K, Mastropaolo J, Deutsch S, Neale JH. PCP and MK-801 induced behaviors reduced by NAAG peptidase inhibition via metabotropic glutamate receptors. E-pub in advance of print, Biological Psychiatry, 2007.

Olszewski RT, Bukhari N, Zhou J, Kozikowski AP, Wroblewski JT, Shamimi-Noori S, Wroblewska B, Bzdega T, Vicini S, Barton FB, Neale JH. NAAG peptidase inhibition reduces locomotor activity and some stereotypes in the PCP model of schizophrenia via group II mGluR. J Neurochem. 2004 May;89(4):876-85. Abstract

View all comments by Joseph Neale

Related News: Studies Explore Glutamate Receptors as Target for Schizophrenia Monotherapy

Comment by:  Dan Javitt, SRF Advisor
Submitted 3 September 2007
Posted 3 September 2007

A toast to success, or new wine in an old skin?
Patil et al. present a landmark study. It is the kind of study that represents the best of how science should work. It pulls together the numerous strands of schizophrenia research from the last 50 years, from the development of PCP psychosis as a model for schizophrenia in the late 1950s, through the links to glutamate, the discovery of metabotropic receptors, and the seminal discovery in 1998 by Moghaddam and Adams that metabotropic glutamate 2/3 receptor (mGluR2/3) agonists reverse the neurochemical and behavioral effects of PCP in rodents (Moghaddam and Adams, 1998. The story would not be possible without the elegant medicinal chemistry of Eli Lilly, which provided the compounds needed to test the theories; the research support of NIMH and NIDA, who have been consistent supporters of the “PCP theory”; or the hard work of academic investigators, who provided the theories and the platforms for testing. The study is large and the effects robust. Assuming they replicate (and there is no reason to suspect that they will not), this compound, and others like it, will represent the first rationally developed drugs for schizophrenia. Patients will benefit, drug companies will benefit, and academic investigators and NIH can feel that they have played their role in new treatment development.

Nevertheless, it is always the prerogative of the academic investigator to ask for more. In this case, we do not yet know if this will be a revolution in the treatment of schizophrenia, or merely a platform shift. What is striking about the study, aside from the effectiveness of LY2140023, is the extremely close parallel in both cross-sectional and temporal pattern of response between it and olanzapine. Both drugs change positive and negative symptoms in roughly equal proportions, despite their different pharmacological targets. Both drugs show approximately equal slopes over a 4-week period. There is no intrinsic reason why symptoms should require 4 or more weeks to resolve, or why negative and positive symptoms should change in roughly the same proportion with two medications from two such different categories, except that evidently they do.

There are many things about mGluR2/3 agonists that we do not yet know. The medication used here was administered at a single, fixed dose. It is possible that a higher dose might have been better, and that optimal results have not yet been achieved. The medications were used in parallel. It is possible that combined medication might be more effective than treatment with either class alone. The study was stopped at 4 weeks, with the trend lines still going down. It is possible that longer treatment duration in future studies might lead to even more marked improvement and that the LY and olanzapine lines might separate. No cognitive data are reported. It is possible that marked cognitive improvement will be observed with these compounds when cognition is finally tested, in which case a breakthrough in pharmacotherapy will clearly have been achieved.

If one were to look at the glass as half empty, then the question is why the metabotropic agonist did not beat olanzapine, and why the profiles of response were so similar. If these compounds work, as suggested in the article by modulating mesolimbic dopamine, then it is possible that metabotropic agonists will share the same therapeutic limitations as current antipsychotics—good drugs certainly and without the metabolic side effects of olanzapine, but not “cures.” The recent study with the glycine transport inhibitor sarcosine by Lane and colleagues showed roughly similar overall change in PANSS total (-17.1 pts) to that reported in this study, but larger change in negative symptoms (-5.5 pts), and less change in positive symptoms (-2.3 pts) in a similar type of patient population. Onset of effect in the sarcosine study also appeared somewhat faster. The sarcosine study was smaller (n = 20) and did not include a true placebo group. As with the Lilly study, it was only 4 weeks in duration, and did not include cognitive measures. It also included only two, possibly non-optimized doses. As medications become increasingly available to test a variety of mechanisms, side-by-side comparisons will become increasingly important.

There are also causes for concern and effects to be watched. For example, a side effect signal was observed for affect lability in the LY group, at about the same prevalence rate as weight increase in the olanzapine group. What this means for the mechanism and how this will effect treatment remains to be determined. Since these medications are agonists, there is concern that metabotropic receptors may downregulate over time. Thus, whether treatment effects increase, decrease, or remain constant over the course of long-term treatment will need to be determined. Nevertheless, 50 years since the near-contemporaneous discovery of both PCP and chlorpromazine, it appears that glutamatergic drugs for schizophrenia may finally be on the horizon.

References:

Moghaddam B, Adams BW. Reversal of phencyclidine effects by a group II metabotropic glutamate receptor agonist in rats. Science. 1998 Aug 28;281(5381):1349-52. Abstract

View all comments by Dan Javitt

Related News: Studies Explore Glutamate Receptors as Target for Schizophrenia Monotherapy

Comment by:  Gulraj Grewal
Submitted 4 September 2007
Posted 4 September 2007
  I recommend the Primary Papers

Related News: Studies Explore Glutamate Receptors as Target for Schizophrenia Monotherapy

Comment by:  Shoreh Ershadi
Submitted 8 June 2008
Posted 9 June 2008
  I recommend the Primary Papers

Related News: ICOSR 2009—Unpleasing Placebos Cloud Antipsychotic Drug Trials

Comment by:  Paul Shepard
Submitted 23 April 2009
Posted 26 April 2009

When the 17 sites with high placebo responders were removed from the analysis, were only participants randomized to placebo removed or were all subjects who were recruited at these sites removed?

View all comments by Paul Shepard

Related News: ICOSR 2009—Unpleasing Placebos Cloud Antipsychotic Drug Trials

Comment by:  C. Anthony Altar
Submitted 28 April 2009
Posted 2 May 2009

Reply to P. Shepard
At ICOSR, Dr. Kinon presented the effects on PANSS positive values over 4 weeks for the placebo group, the groups receiving various LY2140023 doses, and those receiving olanzepine, but "without the 17 sites." I am reasonably sure, but not 100% positive, that this excluded all data from those sites, not just the placebo responders. Anything less would have introduced an unacceptable bias, even for a post-hoc analysis.

View all comments by C. Anthony Altar

Related News: ICOSR 2009—Unpleasing Placebos Cloud Antipsychotic Drug Trials

Comment by:  Ralph Hoffman
Submitted 19 May 2009
Posted 20 May 2009

These placebo results are certainly irksome, but may be important in positive ways. I am thinking of two hypotheses to account for these results. First, perhaps second-generation antipsychotic drugs (that are now more widely in use than ever) have more sustained therapeutic effects after discontinuation, so when patients are taken off their prescribed drugs to participate in these trials, their vulnerability to symptomatic worsening is less.

Of course, this would not explain the greater improvements in placebo groups. But perhaps with growing expectations regarding patient safety and support during randomized clinical trials overall, participants are getting more contact with research staff, which may have non-specific positive effects. We have, for instance, solid data indicating that significant social isolation is a trigger for psychotic symptoms independent of neuropsychological impairment in vulnerable individuals (unpublished data). The combination of reduced social isolation, increased staff support, plus (perhaps) sustained protective effects of second-generation drugs might account for emergence of greater positive placebo response.

View all comments by Ralph Hoffman