Utilizing microarray technology, the present study demonstrates that repeated administration of mGlu5 receptor antagonists MPEP and MTEP produces changes in the expression of a multitude of genes in the rat frontal cortex. The functions of these genes were not confined to synaptic transmission or signal transduction pathways known to be linked to mGlu5 receptor function, but instead represented a host of biological functions including regulation of biosynthesis and metabolism, cell-cell adhesion and communication, cell cycle control, and protein, protein kinase and nucleotide binding activity. In addition, expression of genes involved in immune system function, ion homeostasis and transport, and nervous system development was also altered. Our data suggest that mGluR5 receptors may regulate a host of cellular functions other than modulatory synaptic transmission by glutamate. A statistically significant correlation between the fold-change observed by microarray analysis and the fold-change of 9 genes selected for analysis by qPCR was observed, indicating a degree of reliability in the changes in gene expression as detected by microarray analysis. However, additional studies examining whether these alterations in gene expression result in changes in actual protein synthesis are needed to further confirm these findings.
The doses of MPEP and MTEP chosen for the present study were each 10 mg/kg i.p.. It has previously been shown that this dose of MPEP produces rapid and full occupancy of mGlu5 receptors in the rat brain following systemic administration (Anderson et al., 2003
). In contrast, only a 3 mg/kg i.p. dose of MTEP has been shown to be required to produce full occupancy of brain mGlu5 receptors. However, Anderson and colleagues also noted differences in the clearance rates of MPEP and MTEP from the brain. For example, approximately 4 hours following systemic administration MPEP or MTEP, these investigators found that mGlu5 receptor occupancy had declined to approximately 40-50% in MPEP rats, whereas in MTEP treated rats mGlu5 receptor occupancy had declined to approximately 5-10% (Anderson et al., 2003
). Therefore, while MTEP appears to be a more potent mGlu5 antagonist, it also appears to be shorter acting in the brain. We therefore attempted to compensate for this by increasing the dose of MTEP to 10 mg/kg so as to obtain roughly equal durations of mGlu5 receptor occupancy following each injection. Recent microdialysis studies have shown that extracellular levels of both MPEP and MTEP are in the high nanomolar to low micromolar range following systemic administration of similar doses (Nagel et al., 2007
), and therefore likely retain their selectivity for mGlu5 receptors over other off-target proteins (Cosford et al., 2003
; Gasparini et al., 1999
). However, the receptor occupancy study (Anderson et al., 2003
) and the microdialysis study (Nagel et al., 2007
) were performed following acute administration of MPEP or MTEP, and it is unknown how the receptor occupancy, extracellular levels or pharmacokinetics of MPEP or MTEP are altered following repeated administration twice daily, as was performed in the present study.
Various studies examining the behavioral effects of MPEP and MTEP in animal models of CNS disorders have demonstrated positive effects after acute systemic administration (c.f., Brodkin et al., 2002b
; Chiamulera et al., 2001
; Pietraszek et al., 2005
; Spooren et al., 2000
; Tatarczynska et al., 2001
; Tessari et al., 2004
; Varty et al., 2005
). However, some investigators have reported that multiple dosing procedures with these compounds produces increases efficacy in the anxiolytic, antidepressant or anti-addictive effects as compared with acute administration (Backstrom et al., 2004
; Cowen et al., 2005
; Klodzinska et al., 2004
; Li et al., 2006
; Nordquist et al., 2007
; Pilc et al., 2002
). This raises the possibility that changes in gene expression may underlie some of the behavioral effects of MPEP and/or MTEP following repeated exposure. Thus, a multiple dosing schedule was utilized in the present study to determine effects of these compounds on changes in gene expression.
The expression of potassium voltage-gated channel shaker-related subfamily member 1 (Kcna1
), also termed Kv1.1, was significantly down-regulated by both mGlu5 antagonists, suggesting that these compounds may alter neuronal excitability and activity in the frontal cortex (Homayoun and Moghaddam, 2006
) by modulating potassium channel expression.
Repeated administration of both MPEP and MTEP was found to decrease the expression of numerous signaling proteins that are known to be downstream of mGlu5 receptors, including proteins involved to calcium second messenger and MAPK-related signaling such as CaM kinase II inhibitor alpha (Camk2n1
), protein kinase AMP activated alpha 2 catalytic subunit (Prkaa2
), also termed protein kinase A Cα2
, calcium/calmodulin dependent protein kinase II beta (Camk2b
), also termed CaMKIIβ, and striatin/calmodulin-binding protein 3 (Strn3
) The mGluR5 antagonists also decreased the expression of protein kinase C binding protein 1 (Prkcbp1
) and the predicted sequence of RhoGAP involved in beta-catenin-N-cadherin and NMDA receptor signaling (RICS_pred
). Many of these proteins are postsynaptic molecular substrates that underlie synaptic plasticity, which may explain the deleterious effects of mGlu5 antagonists on some forms of learning and memory (Simonyi et al., 2005
). Despite the well-established fact that mGlu5 receptors are coupled to PKC signaling, we did not observe changes in the expression of any PKC isoforms.
The expression of the gene encoding Casitas B-lineage lymphoma b (Cblb
) was significantly downregulated by both MPEP and MTEP treatment. mGlu5 receptors are constitutively expressed on lymphocytes (Pacheco et al., 2004
), and it is therefore possible that MPEP and MTEP may have immunomodulatory properties, although we are unaware of the existence of any such reports on this topic in the literature.
Both MPEP and MTEP altered the expression of genes encoding proteins involved in biosynthesis such as phosphatidylserine synthase 1 (Ptdss1) and platelet-activating factor acetylhydrolase isoform Ib alpha subunit (Pafah1b1), cell-cell adhesion and intercellular communication proteins such as epidermal growth factor receptor (Egfr) and beta-catenin (Ctnnb1), and cell cycle control proteins such as cyclin D2 (Ccnd2) and the predicted sequence for cyclin T2 (Ccnt2-pred). These compounds also down-regulated the expression of CNS development proteins such as sorting nexin family member 27 (Snx27), and transcription factors and DNA-binding proteins such as zinc finger proteins 422 (Znf422_pred), and 655 (Zfp655), nuclear factor I/B (Nfib), inhibitor of DNA binding 4 (Id4), and eukaryotic translation initiating factor 4E binding protein 2 (Eif4ebp2) and the predicted sequences of eukaryotic translation initiating factor 2C member 1 (Eif2c1_pred) and RNA-binding motif protein 9 (Rbm9_pred). These findings suggest that some of the observed effects of mGlu5 antagonists on gene expression may be mediated by down-regulation of the expression of various transcription factors and nucleotide binding proteins.
Of particular interest was the observation that both MPEP and MTEP decreased the expression of the gene encoding the glutamate transporter solute carrier family 1 member 2 (Slc1a2
), also known as excitatory amino acid transporter 2 (EAAT2) or glutamate transporter 1 (GLT-1). This sodium-dependent glutamate transporter is localized to presynaptic terminals and regulates extracellular levels of glutamate (Shigeri et al., 2004
). One might hypothesize that the ability of repeated administration of MPEP and MTEP to decrease the expression of EAAT2/GLT-1 might result in increased extracellular levels of glutamate. While it has been reported that local application of MPEP in vitro (Thomas et al., 2001
) or in vivo (de Novellis et al., 2003
; Mills et al., 2001
; Thomas et al., 2001
) actually reduces extracellular levels of glutamate, to our knowledge no studies to date have measured changes in extracellular glutamate in response to repeated administration of mGlu5 antagonists, as was performed in the present study.
Pathway analysis revealed that many of the common biological pathways whose components were altered by both MPEP and MTEP were related to biosynthesis (ATP synthesis, hydrolase activity), muscle development, or various intracellular signaling pathways (TGF-beta, Wnt, neuropeptide, and MAPK signaling). Surprisingly, the expression of the gene encoding mGlu5 (Grm5
) was not altered by repeated administration of MPEP or MTEP, and this lack of change in expression was confirmed by qPCR. These data indicate a substantial degree of resilience in expression this receptor in the frontal cortex in response to repeated administration of the antagonists MPEP or MTEP (10 mg/kg each twice daily for 5 days). These data are consistent with that of a previous report, which showed that repeated treatment of MTEP (2 mg/kg/day for 12 days) to rats produced no changes in mGlu5 mRNA expression in the cingulate cortex, dorsal striatum or nucleus accumbens, as measured by in situ hybridization (Cowen et al., 2005
). However, these authors did note a 25% reduction in mGlu5 mRNA expression in the olfactory tubercle induced by repeated MTEP administration, suggesting that the mechanisms regulating the expression of mGlu5 in response to repeated antagonist treatment may be brain region-specific.
In summary, we have demonstrated that repeated administration of the mGlu5 antagonists MPEP and MTEP resulted in changes in the expression of 63 genes, with a predominant trend towards inhibition of gene expression since 58 genes were down-regulated and only 5 were up-regulated. Genes whose expression was altered by these mGluR5 antagonists encode proteins known to be involved in biosynthesis and metabolism, cell-cell adhesion and communication, cell cycle control, immune system function, ion homeostasis and transport, nervous system development, transcription factors and DNA-binding proteins, protein kinase or phosphatase activity, protein synthesis, modification and trafficking, signal transduction, and synaptic transmission. These data suggest that mGlu5 antagonists can alter the expression of a wide array of genes that is much larger than previously characterized. Given that mGlu5 receptors are coupled to numerous signal transduction cascades, and are expressed on cells of the immune system, we speculate that ability of MPEP and MTEP to alter the expression of genes related to intracellular signaling cascades (i.e., MAPK) and immune system function are primarily related to the pharmacological actions of these compounds. In contrast, given the observed changes in expression of numerous genes not previously associated with mGlu5 function (i.e., biosynthesis and metabolism, cell-cell adhesion, and cell cycle control), we speculate that these changes may be secondary to the ability of these compounds to alter gene expression via specific transcription factors or nucleotide binding proteins.