A growing body of data shows that the AMPA subtype of glutamate receptors play a major role in regulating short and long term forms of synaptic plasticity. Furthermore, it is now clear that regulation of plasticity occurs predominantly by regulating the trafficking of AMPA receptor subunits, and their insertion and removal from the synapse. Notably, this trafficking is primarily dependent upon AMPA receptor subunit phosphorylation by 3 major signaling pathways known to be targets for mood stabilizers: the PKC, PKA and MAPK cascades. In view of the growing body of data suggesting that severe mood disorders may be associated with impairments of cellular plasticity, we undertook the present series of studies to determine if two clinically effective, but structurally highly dissimilar antimanic agents, lithium and VPA, regulate synaptic expression of AMPA receptor subunit GluR1. Administration of chronic lithium or valproate (at therapeutically relevant concentrations) reduced rat hippocampal synaptosomal levels of GluR1 after by 40% and 20%, respectively. In cultured hippocampal neurons, both lithium and VPA significantly down regulated the surface expression of GluR1 ~ 40% in a dose and time-dependent manner. Surface staining with an anti-N terminal GluR1 antibody confirmed the result. Double-immunostaining of GluR1 and synaptotagmin showed that chronic treatment attenuated the numbers of GluR1 positive synapses of lithium and valproate-treated neurons. However, total protein levels of GluR1, and synaptotagmin remained unchanged after lithium and valproate treatment in vitro and in vivo. Lithium and valproate treatment also attenuated the phosphorylation of a specific PKA site (GluRp845) by 52 and 33% respectively. Sp-cAMP treatment reversed the attenuation of phosphorylation by lithium and valproate and also brought GluR1s back to the surface, suggesting that phosphorylation of GluRp845 is involved in the mechanism of GluR1 surface attenuation. In striking contrast, drugs, such as imipramine, which induce mania, increase the synaptic expression of GluR1 in vivo in hippocampus. These studies suggest that regulation of glutamatergically mediated synaptic plasticity may play a role in the treatment of mood disorders, and raises the possibility that agents more directly affecting synaptic GluR1 may represent novel therapies for this devastating illness. In order to develop a new potential drug which mimics the effect of mood stabilizers on GluR1 phosphorylation, TAT-peptides (TAT-p845 and TAT-SRC) were designed and synthesized. Tat peptide (YGRKKRRQRRR) is a leading peptide, which enables the functional peptide to pass through the blood brain barrier and cell membrane, allowing it to get into cytosol or synapses of the neurons. A previous study has successfully utilized peptide injection into animals to disrupt the interaction of PSD-95 with NMDA receptors in the brain and to provide a neuroprotective effect on a stroke animal model. TAT-p845 was able to inhibit the phosphorylation of AMPA receptors at its PKA site and down-regulate the surface expression of GluR1 in cultured hippocampal neurons, which is the same effect produced by lithium and valproate. Moreover, this TAT-p845 was able to pass the blood brain barrier and inhibit the phosphorylation of GluR1 in the hippocampus in vivo, which again demonstrated its ability to induce the same effects as lithium and valproate. On going behavioral studies should determine the effects of this peptide on mood associated behavior. TAT-p845, which attenuates AMPA receptor levels at synapses, may offer exciting possibilities as a new class of medicine with the potential for treatment of: bipolar disorder. We also investigated the effects of mood stabilizers on GluR2 synaptic expression/trafficking. The GluR2 is known to regulate calcium permeability, rectification, and single channel conductance of AMPA receptors; notably, these receptors are dominantly influenced by inclusion of a GluR2 subunit in the receptor complex, suggesting that alterations in synaptic GluR2 trafficking would have a major effect on synaptic plasticity. Rats were treated with lithium, valproate, or imipramine for 4 weeks and synaptosomal fractions from the hippocampus were prepared. Western blot analysis with an anti-GluR2 antibody was performed to determine the GluR2 content at synapses and in the brain. In order to understand the underlining mechanism, GluR-2 expression at the neuronal surface were determined by biotinylation assay and GluR-2 levels at the synapses was investigated by double immunostaining with anti-GluR2 and anti-synaptotagmin antibodies in cultured hippocampal neurons. We found that both lithium and valproate reduced hippocampal synaptosomal levels of GluR2 after chronic administration. Lithium significantly reduced GluR2 total protein levels in chronically treated animals, while valproate did not. In cultured hippocampal neurons, both lithium and VPA significantly down regulated the surface expression of GluR 2 in a time-dependent manner. Double-immunostaining of GluR2 and synaptotagmin showed that the GluR2 immunostaining at synapses of lithium and valproate-treated neurons was attenuated after 4 days of treatment. Lithium, but not valproate, significantly attenuated total protein levels of GluR2, confirming the in vivo data. In striking contrast, mania inducing drugs, such as imipramine, increase the synaptic expression of GluR2 in vivo in hippocampus. These studies suggest that regulation of glutamatergically mediated synaptic plasticity may play a role in the treatment of mood disorders, and raises the possibility that agents more directly affecting synaptic GluR2 may represent novel therapies for this devastating illness. To further test the hypothesis, we investigated the other antidepressant agents, such as lamotrigine and riluzole, which have efficacy in the depressive phase of bipolar disorder. We sought to determine the role of these anticonvulsants compared to valproate on AMPA receptor trafficking. Hippocampal neurons (10 DIV) were treated with therapeutically relevant concentrations of lamotrigine (20uM), riluzole (2uM) and valproate (1.0mM) for 3 days. Surface GluR1 levels and phosphorylation of GluR1 at p845 were determined. We found that the agents with a predominantly antidepressant profile, namely lamotrigine and riluzole, significantly enhanced surface expression of GluR1 by 152%, 208% and GluR2 by 153% and 128%, respectively. By contrast, the predominantly antimanic agent valproate significantly reduced surface expression of GluR1 and GluR2. Phosphorylation of GluR1 at PKA site was enhanced in both lamotrigine- and riluzole- treated hippocampal neurons, but reduced in valproate treated neurons. Total GluR1 expression enhanced in riluzole-treated neurons, however, remained unchanged in lamotrigine- and valproate- treated neurons. Our findings suggest that GluR1/2 trafficking may be responsible for the different clinical profile of anticonvulsants (antimanic or antidepressant) and suggest avenues for the development of novel therapeutics for these illnesses.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Intramural Research (Z01)
Project #
1Z01MH002831-03
Application #
7137866
Study Section
(LMP)
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2005
Total Cost
Indirect Cost
Name
U.S. National Institute of Mental Health
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Catapano, Lisa A; Manji, Husseini K (2008) Kinases as drug targets in the treatment of bipolar disorder. Drug Discov Today 13:295-302
Catapano, Lisa A; Manji, Husseini K (2007) G protein-coupled receptors in major psychiatric disorders. Biochim Biophys Acta 1768:976-93
Du, Jing; Suzuki, Katsuji; Wei, Yanling et al. (2007) The anticonvulsants lamotrigine, riluzole, and valproate differentially regulate AMPA receptor membrane localization: relationship to clinical effects in mood disorders. Neuropsychopharmacology 32:793-802
DU, Jing; Quiroz, Jorge; Yuan, Peixiong et al. (2004) Bipolar disorder: involvement of signaling cascades and AMPA receptor trafficking at synapses. Neuron Glia Biol 1:231-43