Schizophrenia Research Forum - A Catalyst for Creative Thinking

SfN Atlanta: Working Both Sides of the Synapse in Mood Disorders

Editor's Note: In the second of her meeting missives from the Neurosciences 2006 meeting in Atlanta, Susannah F. Locke of the University of Pennsylvania reports on a symposium entitled, “Protein Trafficking at Synapses and Therapeutic Agents for Mood Disorders.” Co-chaired by Maurizio Popoli of the University of Milan and Mark Rasenick of the University of Illinois, the session covered the regulation of both presynaptic and postsynaptic trafficking in the context of pharmacotherapy for mood disorders.

Regulation of NET by CamKII and by ubiquitination
14 November 2006. Uhna Sung, of Vanderbilt University, gave a presentation entitled “Protein Complexes to Support Trafficking of Antidepressant-sensitive Norepinephrine Transporters.” She discussed the regulation of norepinephrine transporters (NETs) by calcium/calmodulin-dependent protein kinases I and II (CaMKI and -II) and by ubiquitination. CaMKI and -II and E3 ubiquitin ligase Nedd4 were identified from a NET-associated proteome study using CAD (a noradrenergic neuroblastoma line) cells exogenously expressing NET. Sung had previously used the NET proteome to demonstrate associations with both protein phosphatase 2A and 14-3-3 (Sung et al., 2005).

Sung presented data implicating both CaMKI and -II in calcium-dependent NET trafficking. CaMKI and -II siRNA expression in CAD-NET cells attenuated calcium-dependent NET trafficking. CaMKII siRNA expression in CAD-NET cells also decreased NET on the cell surface. Depletion of calcium produced a similar effect which was dependent on an amino-terminus domain of NET. Although Sung did not include this evidence in her talk, she and her collaborators have shown that amphetamine reduces NET localization on the cell surface in a CaMKII-dependent manner (Dipace et al., 2006).

Sung also identified many ubiquitin system enzymes in the NET proteome, including E3 ubiquitin ligase Nedd4. Ubiquitination is a cellular process for tagging proteins for degradation. Immunoprecipitation of NET from CAD cells revealed interactions of NET with Nedd4, and immunofluorescence microscopy demonstrated that Nedd4 and NET colocalize in neuronal processes of cultured primary sympathetic neurons. Treatment with the antidepressant desipramine (a norepinephrine reuptake inhibitor) decreased the ubiquitination of NET and increased total protein levels of NET.

AMPA trafficking in vivo
Jing Du’s talk, “Modulation of AMPA Receptor Trafficking by Mood Stabilizers: Involvement in Antimanic Effects in Animal Models,” focused on administration of a TAT-tagged peptide to modify AMPA trafficking in vivo. Du, of the National Institute of Mental Health, has previously published work exploring the regulation of AMPA glutamate receptor subunit 1 (GluR1) by anti-manic drugs (Du et al., 2004). In this paper he demonstrated that chronic lithium or valproate treatment in rats decreased GluR1 at the synapse and decreased GluR1 phosphorylation at its PKA site. In vitro, these effects could be reversed by treatment with the PKA activator Sp-cAMP.

Du presented new in vivo data demonstrating that a TAT-tagged peptide had effects similar to lithium and valproate in mice. The peptide included the PKA phosphorylation motif of GluR1 and a TAT tag (to ensure delivery across the blood-brain barrier). After several days of peptide injections, mice displayed a decrease in synaptic GluR1 and GluR2. Du also shared preliminary research suggesting that the peptide interferes with amphetamine-induced behavior. Peptide treatment seemed to impact two animal models of manic behavior, decreasing amphetamine-induced hyperactivity and reducing amphetamine association in conditioned place preference tests.

Antidepressants and glutamate release
Maurizio Popoli, of the University of Milan, gave a presentation entitled “Presynaptic Protein Interactions Regulating Glutamate Release in the Action of Stress and Antidepressants.” The majority of his talk covered previously published work on antidepressants (Bonanno et al., 2005).

Popoli described his studies using synaptosomes (purified synaptic terminals) prepared from rats chronically treated with antidepressants. Rats that were administered fluoxetine, reboxetine, or desipramine had similar changes in glutamate release, even though these antidepressants work through different mechanisms. Synaptosomes from antidepressant-treated rats displayed a decrease in depolarization-evoked, but not ionomycin-evoked, glutamate release. GABA release was not affected, suggesting a shift in excitatory versus inhibitory neurotransmission.

Popoli then discussed other studies in which synaptosomes were purified further into a synaptic membrane fraction (which contained the readily releasable pool of vesicles) and a total synaptic vesicle fraction. Synaptic membranes from rats treated with fluoxetine or reboxetine had lower expression of the SNARE proteins syntaxin, SNAP-25, and synaptobrevin. In addition, syntaxin I availability for the SNARE complex was likely inhibited due to changes in protein-protein interactions. Synaptic membranes from treated rats showed less interaction between syntaxin I and alpha-CaM kinase II, but more interaction between syntaxin I and Munc-18. Alpha-CaM kinase II is thought to increase, whereas Munc-18 is thought to decrease syntaxin I’s assembly into the SNARE complex. More recent research in the Popoli lab includes explorations of glutamate release modulation in animal models of depression and stress.

Depression and G protein signaling
Robert Donati, of the Illinois College of Optometry, gave a talk entitled “G Protein Signaling in Microdomains of the Plasma Membrane Is Altered in Depression or by Antidepressant Treatment.” Various types of antidepressant therapy, including electroconvulsive shock therapy and a wide range of antidepressant drugs, increase coupling between Gs-alpha and adenylyl cyclase.

Donati studied the membrane localization of Gs-alpha in Triton X-100 soluble and Triton X-100 insoluble fractions, the latter of which includes both lipid rafts and caveolae. Localization in lipid rafts/caveolae essentially inhibits Gs-alpha by separating it from G-protein coupled receptors and by enhancing its internalization. Donati explained that B-adrenergic signaling in C6 glioma cells causes Gs-alpha to internalize. This internalization is lost when the lipid rafts/caveolae are chemically disrupted (Allen et al., 2005).

Donati presented data exploring the effects of antidepressants on Gs-alpha localization (Donati and Rasenick, 2005). In C6 glioma cells, chronic desipramine or fluoxetine treatment shifted a large percentage of Gs-alpha from the lipid rafts/caveolae fraction to the Triton X-100 soluble fraction. Microtubule disruption created the same pattern of localization, which suggests that microtubules help anchor Gs-alpha in lipid rafts/caveolae.

Donati also shared newer work on human patients. Postmortem studies on depressed patients that had committed suicide showed that there was more Gs-alpha associated with lipid rafts/caveolae in cortex and in cerebellum than in controls.—Susannah F. Locke.

Comments on Related News

Related News: Glutamate Receptor Trafficking—AMPARs Rooted by Stargazin

Comment by:  Monica Beneyto
Submitted 8 March 2007
Posted 10 March 2007
  I recommend the Primary Papers

These two very interesting papers, a welcome article testing previous models proposed for the role of stargazin in AMPA receptor trafficking and a mini-review about TARPs, represent a much needed update on AMPA receptor trafficking and offer some insight into the possible effect disturbances of these proteins have in schizophrenia.

As reported by Tom Fagan, several lines of evidence have led to the current thinking that disruptions in any of the numerous mechanisms that influence the function of NMDA receptors, by either modifying the kinetics of the NMDA channel itself, or the function of proteins that link NMDA receptors to signal transduction pathways, can reproduce schizophrenic symptoms in normal subjects (see related SRF current hypothesis by Bita Mohaddam). The NMDA receptor dysfunction hypothesis of schizophrenia has subsequently been refined to include abnormalities of other glutamate receptor subtypes (AMPA, kainate, and metabotropic receptors), glutamate transporters, and glutamatergic enzymes. Glutamate has a central role in behaviors such as associative learning, working memory, and attention that are often impaired in this illness. Underlying these behaviors are electrophysiological events, such as long-term potentiation, that are mediated in many brain regions by NMDA and AMPA receptor colocalization, activation and signaling. AMPA receptor colocalization with NMDA receptors is crucial for NMDA receptor function, as sustained activation of nearby AMPA receptors is necessary to depolarize the postsynaptic cell and release the tonic Mg2+ blockade of the NMDA receptor, permitting NMDA receptor activation. It is in this scenario where the role of the protein stargazin seems to be crucial. Clinical studies have suggested that ampakines, AMPA receptor positive modulators, can improve cognitive function in schizophrenia, while enhancement of AMPA receptor-mediated currents by these compounds may potentiate the efficacy of antipsychotics (Coyle, 1996). These pharmacologic observations, taken together with a central role for AMPA receptor function in the facilitation of the molecular events that likely underlie learning, memory, and attention, suggest that AMPA receptors may be abnormal in schizophrenia.

AMPA receptor localization, cell surface expression, and activity-dependent modulation are regulated and maintained by a complex network of protein-protein interactions, including those involving stargazin, associated with targeting, anchoring, and spatially organizing synaptic proteins at the cell membrane. These proteins alter receptor sensitivity to glutamate and modulate signaling cascades associated with synaptic transmission by linking AMPA receptors to critical intracellular effector molecules. AMPA receptor trafficking as a critical aspect in the regulation of synaptic efficacy has been recently reviewed (Barry and Ziff, 2002; Malinow, 2003; Malinow and Malenka, 2002; Scannevin and Huganir, 2000; Sheng, 1997; Sheng, 2001; Sheng and Hyoung Lee, 2003; Sheng and Kim, 2002; Sheng and Lee, 2001; Sheng and Nakagawa, 2002; Sheng and Pak, 1999).

Trafficking of AMPA receptors in the postsynaptic cell occurs in several integrated pathways. The constitutive pathway consists of a basal continuous and rapid recycling of GluR2-containing AMPA receptors from non-synaptic to synaptic sites. It has been suggested that NSF [N-ethylmaleimide-sensitive factor] regulates this pool of AMPA receptors independently of NMDA activation by its specific binding to GluR2 (Song et al., 1998). The regulatory pathway, unlike the constitutive pathway, is activity-dependent, triggered by NMDA receptor activation, and driven by PDZ interactions between C-terminal regions of the GluR2 and GluR3 AMPA subunits with the intracellular proteins PICK1, GRIP1, and ABP (Chung et al., 2000; Dev et al., 1999a; Hirbec et al., 2002; Kim et al., 2001; Lu and Ziff, 2005; Osten et al., 2000; Perez et al., 2001; Terashima et al., 2004; Wyszynski et al., 1999; Xia et al., 1999). The new synthesis pathway is dependent on an increase in glutamate activation in the synapse and involves the GluR1 AMPA subunit. Finally, a fourth pathway is mediated by the protein stargazin, the star of these two recent Neuron papers, that binds to all four AMPA subunits and has been identified as a mediator of AMPA receptor function in a two-step trafficking model, in which it first conveys AMPA receptors to the neuronal surface and then sweeps them laterally into postsynaptic sites (Chen et al., 2000). As demonstrated by Bats and colleagues, this process requires an interaction of the carboxy terminus of stargazin with a PDZ anchoring protein such as PSD95, that regulates the diffusion and mediates the clustering of AMPA with NMDA receptors.

We and others have systematically examined the expression of the AMPA receptors, and other molecules associated with glutamate neurotransmission, in postmortem brain samples from persons with schizophrenia. We have previously examined both NMDA and AMPA receptors in multiple brain regions, and have typically found only modest changes in both transcript and protein levels (Clinton et al., 2003; Clinton and Meador-Woodruff, 2004a; Clinton and Meador-Woodruff, 2004b; Healy et al., 1998a; Ibrahim et al., 2000b; Kristiansen and Meador-Woodruff, 2005; Meador-Woodruff et al., 1996; Meador-Woodruff et al., 2003; Meador-Woodruff and Healy, 2000; Meador-Woodruff et al., 2001; Mueller and Meador-Woodruff, 2004). Given the strength of clinical and pharmacological data suggesting glutamate receptor dysfunction in schizophrenia, our focus then shifted to studying intracellular signaling pathways associated with the ionotropic glutamate receptors, postulating that a receptor abnormality might not be a problem with the receptor itself, but rather with associated intracellular trafficking mechanisms.

Several studies have been conducted in vitro to characterize these molecules and their interactions, but few studies have demonstrated their expression in brain, particularly in regions such as the hippocampus, cerebellum, and cortex (Puschel et al., 1994; Chen et al., 2000). Our report in the Journal of Comparative Neurology two years ago showed the distribution of transcripts encoding stargazin, and another five intracellular proteins related to the AMPA receptors in the macaque brain. In our study stargazin was highly expressed in some regions of the hippocampus, thalamus, pons, cerebellum and specially in the cerebral cortex (Beneyto and Meador-Woodruff, 2004).

In a recent paper published in Synapse we identified abnormalities in the expression of transcripts encoding the proteins PICK1 and stargazin in the dorsolateral prefrontal cortex (DLPFC) in schizophrenia (Beneyto and Meador-Woodruff, 2006). Results from in situ hybridization experiments showed lamina-specific decreased expression of PICK1 transcripts and increased expression of stargazin mRNA in DLPFC in layer III in schizophrenia. Recent reports have demonstrated PICK1 involvement in exocytosis of GluR2-containing receptors into the plasma membrane in cerebellar stellate cells (Gardner et al., 2005; Liu and Cull-Candy, 2005; Malinow and Malenka, 2002). Decreased expression of PICK1 suggests increased number of AMPA receptors available to bind to ABP/GRIP and thereby be sequestered in immobile vesicles inside the PSD not ready to be inserted at the postsynaptic membrane, resulting in decreased number of AMPA receptors at the cell surface in schizophrenia. In this scenario, the increased stargazin expression we found could be a compensation for the decreased number of AMPA receptors inserted at the postsynaptic membrane.

Stargazin is responsible for lateral transport of AMPA receptors already inserted in the membrane from extrasynaptic to synaptic sites and for their colocalization with NMDA receptors (Tomita et al., 2004, 2005; Turetsky et al., 2005). Perhaps more importantly, stargazin also acts as a positive allosteric modulator of AMPA receptor ion channel function. Recent data suggest that AMPA receptors complexed with stargazin at the postsynaptic membrane are significantly more responsive to synaptically released glutamate, compared with AMPA receptors lacking a stargazin interaction (Priel et al., 2005; Tomita et al., 2005; Yamazaki et al., 2004). Coexpression of stargazin with AMPA receptor subunits significantly reduces receptor desensitization in response to glutamate, slowing receptor deactivation rates and accelerating the recovery from desensitization. Structurally, based on this tight correlation between desensitization and the stability of the AMPA receptor intradimer interface, binding of stargazin has been suggested to be a stabilizer of receptor conformation (Priel et al., 2005). This fact, together with the AMPA receptor trafficking role of stargazin, suggests that the stargazin upregulation we found may be secondary to decreased PICK1 expression and possible reduced AMPA receptor membrane expression in DLPFC in schizophrenia.

Regulated insertion and removal of AMPA receptors at the synapse provides a mechanism for altering synaptic efficacy (Malenka and Nicoll, 1999; Malinow et al., 2000). Taken together, changes of AMPA subunit and related protein expression in the prefrontal cortex of schizophrenic patients might provide a molecular basis for both structural and neurochemical impairments in this illness.

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Related News: DISC1 and SNAP23 Emerge In NMDA Receptor Signaling

Comment by:  Jacqueline Rose
Submitted 2 March 2010
Posted 2 March 2010
  I recommend the Primary Papers

The newly published paper by Katherine Roche and Paul Roche reports SNAP-23 expression in neuron dendrites and examines the possible role of this neuronal SNAP-23 protein. To this point, SNAP-23 has traditionally been discussed in reference to vesicle trafficking in epithelial cells (see Rodriguez-Boulan et al., 2005 for review), so it is of interest to determine the function of SNAP-23 in neurons. Suh et al. report that surface NMDA receptor expression and NMDA-mediated currents are inhibited following SNAP-23 knockdown. Further, SNAP-23 knockdown results in a specific decrease in NR2B subunit insertion; previously, the NR2B subunit has been reported to preferentially localize to recycling endosomes compared to NR2A (Lavezzari et al., 2004). Given these findings, it is reasonable to conclude that SNAP-23 may be involved in maintaining NMDA receptor surface expression possibly by binding to NMDA-specific recycling endosomes.

Interestingly, there is recent evidence that PKC-induced NMDA receptor insertion is mediated by another neuronal SNARE protein; postsynaptic SNAP-25 (Lau et al., 2010). It is possible that activity-induced NMDA receptor trafficking is mediated by SNAP-25, while baseline maintenance of NMDA receptor levels relies on SNAP-23. Other evidence to suggest a strictly regulatory role for SNAP-23 in neuronal NMDA insertion is the finding that activity-dependent receptor insertion from early endosomes has previously been reported to be restricted to AMPA-type glutamate receptors (Park et al., 2004). However, it is possible that activity-induced insertion of AMPA receptors occurs via a distinct endosome pool than NMDA receptors; AMPA and NMDA receptor trafficking has been reported to proceed by distinct vesicle trafficking pathways (Jeyifous et al., 2009).

Although SNAP-23 may not be involved in activity-dependent early endosome receptor trafficking, it is possible that SNAP-23 operates in other pathways linked to activity-induced NMDA receptor trafficking. For instance, SNAP-23 may be the SNARE protein by which lipid raft shuttling of NMDA receptors occurs. SNAP-23 has been found to preferentially associate with lipid rafts over SNAP-25 in PC12 cells (Salaün et al., 2005). As well, NMDA receptors have been found to associate with lipid raft associated proteins flotilin-1 and -2 in neurons (Swanwick et al., 2009). Lipid raft trafficking of NMDA receptors to post-synaptic densities has been reported to follow global ischemia (Besshoh et al., 2005), and the possibility remains that under certain circumstances, NMDA trafficking occurs by lipid raft association to SNAP-23.

Taken together, the discovery of post-synaptic SNARE proteins offers several avenues of research to determine their roles and functions in glutamatergic synapse organization. Further, investigating disruption of synaptic receptor organization presents several possibilities for potential etiologies of disorders linked to compromised glutamate signaling like schizophrenia.


Besshoh, S., Bawa, D., Teves, L., Wallace, M.C. and Gurd, J.W. (2005). Increased phosphorylation and redistribution of NMDA receptors between synaptic lipid rafts and post-synaptic densities following transient global ischemia in the rat brain. Journal of Neurochemistry, 93: 186-194. Abstract

Jeyifous, O., Waites, C.L., Specht, C.G., Fujisawa, S., Schubert, M., Lin, E.I., Marshall, J., Aoki, C., de Silva, T., Montgomery, J.M., Garner, C.C. and Green, W.N. (2009). SAP97 and CASK mediate sorting of NMDA receptors through a previously unknown secretory pathway. Nature Neuroscience, 12: 1011-1019. Abstract

Lau, C.G., Takayasu, Y., Rodenas-Ruano, A., Paternain, A.V., Lerma, J., Bennet, M.V.L. and Zukin, R.S. (2010). SNAP-25 is a target of protein kinase C phosphorylation critical to NMDA receptor trafficking. Journal of Neuroscience, 30: 242-254. Abstract

Lavezzari, G., McCallum, J., Dewey, C.M. and Roche, K.W. (2004). Subunit-specific regulation of NMDA receptor endocytosis. Journal of Neuroscience, 24: 6383-6391. Abstract

Park, M., Penick, E.C., Edward, J.G., Kauer, J.A. and Ehlers, M.D. (2004). Recycling endosomes supply AMPA receptors for LTP. Science, 305: 1972-1975. Abstract

Rodriguez-Boulan, E., Kreitzer, G. and Müsch, A. (2005) Organization of vesicular trafficking in epithelia. Nature Reviews: Molecular Cell Biology, 6: 233-247. Abstract

Salaün, C., Gould, G.W. and Chamberlain, L.H. (2005). The SNARE proteins SNAP-25 and SNAP-23 display different affinities for lipid rafts in PC12 cells. Journal of Biological Chemistry, 280: 1236-1240. Abstract

Suh, Y.H., Terashima, A., Petralia, R.S., Wenthold, R.J., Isaac, J.T.R., Roche, K.W. and Roche, P.A. (2010). A neuronal role for SNAP-23 in postsynaptic glutamate receptor trafficking. Nat Neurosci. 2010 Mar;13(3):338-43. Abstract

Swanwick, C.C., Shapiro, M.E., Chang, Y.Z. and Wenthold, R.J. (2009). NMDA receptors interact with flotillin-1 and -2, lipid raft-associated proteins. FEBS Letters, 583: 1226-1230. Abstract

View all comments by Jacqueline Rose