Melanie J. Robbins

Pharmacological and immunohistochemical characterization of the APJ receptor and its endogenous ligand apelin

Apelin peptides have recently been identified to be the endogenous ligands for the G protein‐coupled receptor APJ. However, little is known about the physiological roles of this ligand‐receptor pairing. In the present study we investigated the pharmacology of several apelin analogues at the human recombinant APJ receptor using radioligand binding and functional assays. This has led to the identification of key residues in the apelin peptide required for functional potency and binding affinity through structure–activity studies. In particular, we have identified that replacement of leucine in position 5, or arginine in position 2 and 4 of the C‐terminal apelin peptide, apelin‐13, resulted in significant changes in pharmacology. We also investigated the detailed localization of pre‐proapelin and APJ receptor mRNA in a wide range of human, rat and mouse tissues using quantitative RT–PCR, and carried out a detailed immunohistochemical study of the distribution of the APJ receptor in rat brain and spinal cord. Interestingly, the APJ receptor was not only co‐localized in white matter with GFAP in the spinal cord, but was also clearly localized on neurones in the brain, suggesting that this receptor and its peptide may be involved in a wide range of biological process yet to be determined.

The use of TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models

TaqMan reverse transcription polymerase chain reaction (RT-PCR) is a recently developed technique which allows the measurement of an accumulating PCR product in real time. In the present study we have validated the use of TaqMan RT-PCR for mRNA localisation studies in human and rat tissues, and for the investigation of gene expression changes in CNS animal models. In human brain, D(2) receptor mRNA was enriched in caudate nucleus and putamen, whilst in rat brain, highest levels of D(2) receptor mRNA expression were observed in striatum and nucleus accumbens, consistent with the known distribution of this receptor in basal ganglia. In a rat model of permanent middle cerebral artery occlusion (pMCAO), endogenous interleukin-1 receptor antagonist (IL-1ra) mRNA was upregulated over 30-fold at 24 h post-lesion in both striatum and cortex ipsilateral to artery occlusion. Brain-derived neurotrophic factor (BDNF) mRNA was transiently upregulated 3.7-fold at 3 h, but not at 24 h or 3 days after induction of cortical spreading depression (CSD) in rats. Our observations in these two animal models using TaqMan RT-PCR were consistent with previous reports using other techniques. In conclusion, TaqMan RT-PCR assays provide a rapid and reliable method for semi-quantitative analysis of gene expression in the nervous system.

Epileptogenesis and Enhanced Prepulse Inhibition in GABA B1 -Deficient Mice

The recent cloning of two GABA(B) receptor subunits, GABA(B1) and GABA(B2), has raised the possibility that differences in GABA(B) receptor subunit composition may give rise to pharmacologically or functionally distinct receptors. If present, such molecular diversity could permit the selective targeting of GABA(B) receptor subtypes specifically involved in pathologies such as drug addiction, spasticity, pain, and epilepsy. To address these issues we have developed a GABA(B1) subunit knockout mouse using gene targeting techniques. In the brains of GABA(B1) null mice, all pre- and postsynaptic GABA(B) receptor function was absent demonstrating that the GABA(B1) subunit is essential for all GABA(B) receptor-mediated mechanisms. Despite this, GABA(B1) null mice appeared normal at birth, although by postnatal week four their growth was retarded and they developed a generalized epilepsy that resulted in premature death. In addition, GABA(B1) heterozygote animals showed enhanced prepulse inhibition responses compared to littermate controls, suggesting that GABA(B1) deficient mice exhibit increased sensorimotor gating mechanisms. These data suggest that GABA(B) receptor antagonists may be of benefit in the treatment of psychiatric and neurological disorders in which attentional processing is impaired.

The expression of GABAB1 and GABAB2 receptor subunits in the CNS differs from that in peripheral tissues

GABA(B) receptors are G-protein-coupled receptors that mediate the slow and prolonged synaptic actions of GABA in the CNS via the modulation of ion channels. Unusually, GABA(B) receptors form functional heterodimers composed of GABA(B1) and GABA(B2) subunits. The GABA(B1) subunit is essential for ligand binding, whereas the GABA(B2) subunit is essential for functional expression of the receptor dimer at the cell surface. We have used real-time reverse transcriptase-polymerase chain reaction to analyse expression levels of these subunits, and their associated splice variants, in the CNS and peripheral tissues of human and rat. GABA(B1) subunit splice variants were expressed throughout the CNS and peripheral tissues, whereas surprisingly GABA(B2) subunit splice variants were neural specific. Using novel antisera specific to individual GABA(B) receptor subunits, we have confirmed these findings at the protein level. Analysis by immunoblotting demonstrated the presence of the GABA(B1) subunit, but not the GABA(B2) subunit, in uterus and spleen. Furthermore, we have shown the first immunocytochemical analysis of the GABA(B2) subunit in the brain and spinal cord using a GABA(B2)-specific antibody. We have, therefore, identified areas of non-overlap between GABA(B1) and GABA(B2) subunit expression in tissues known to contain functional GABA(B) receptors. Such areas are of interest as they may well contain novel GABA(B) receptor subunit isoforms, expression of which would enable the GABA(B1) subunit to reach the cell surface and form functional GABA(B) receptors.

Comparative immunohistochemical localisation of GABAB1a, GABAB1b and GABAB2 subunits in rat brain, spinal cord and dorsal root ganglion

GABA(B) receptors are G-protein-coupled receptors mediating the slow onset and prolonged synaptic actions of GABA in the CNS. The recent cloning of two genes, GABA(B1) and GABA(B2), has revealed a novel requirement for GABA(B) receptor signalling. Studies have demonstrated that the two receptor subunits associate as a GABA(B1)/GABA(B2) heterodimer to form a functional GABA(B) receptor. In this study we have developed polyclonal antisera specific to two splice variants of the GABA(B1) subunit, GABA(B1a) and GABA(B1b), as well as an antiserum to the GABA(B2) subunit. Using affinity-purified antibodies derived from these antisera we have mapped out the distribution profile of each subunit in rat brain, spinal cord and dorsal root ganglion. In brain the highest areas of GABA(B1a), GABA(B1b) and GABA(B2) subunit expression were found in neocortex, hippocampus, thalamus, cerebellum and habenula. In spinal cord, GABA(B1) and GABA(B2) subunits were expressed in the superficial layers of the dorsal horn, as well as in motor neurones in the deeper layers of the ventral horn. GABA(B) receptor subunit immunoreactivity in dorsal root ganglion suggested that expression of GABA(B1b) was restricted to the large diameter neurones, in contrast to GABA(B1a) and GABA(B2) subunits which were expressed in both large and small diameter neurones. Although expression levels of GABA(B1) and GABA(B2) subunits varied we found no areas in which GABA(B1) was expressed in the absence of GABA(B2). This suggests that most, if not all, GABA(B1) immunoreactivity may represent functional GABA(B) receptors. Although our data are in general agreement with functional studies, some discrepancies in GABA(B1) subunit expression occurred with respect to other immunohistochemical studies. Overall our data suggest that GABA(B) receptors are widely expressed throughout the brain and spinal cord, and that GABA(B1a) and GABA(B1b) subunits can associate with GABA(B2) to form both pre- and post-synaptic receptors.

Comparison between intraperitoneal and subcutaneous phencyclidine administration in Sprague–Dawley rats: A locomotor activity and gene induction study

In a putative model of acute phencyclidine (PCP)-induced psychosis we evaluated effects of the drug on locomotor activity (LMA) and immediate early gene (IEG) induction in the rat using two routes of drug administration, intraperitoneal (i.p.) and subcutaneous (s.c.). Adult male rats received saline or PCP (1.0-5.0 mg/kg) either i.p or s.c. and were assessed for LMA for 60 min. At the end of the LMA testing animals were culled and blood and brain samples were collected for PCP concentration analysis. Separate cohorts of animals received 5.0 mg/kg PCP (i.p. or s.c.) and were used to investigate (1) the pharmacokinetics of PCP or (2) induction of IEG (Arc, c-fos, BDNF, junB, Krox-20, sgk-1, NURR1, fra-2, Krox-24, and egr-3) mRNA expression in the prefrontal cortex (PFC). Administration of PCP resulted in locomotor hyperactivity which was more robust and longer-lasting in animals dosed s.c. compared to i.p.-treated-animals. Differences in hyperlocomotion were paralleled by higher concentrations of PCP in the blood and in the brain of s.c.-treated animals compared to i.p.-treated animals. The differences in the concentration of PCP between the two routes of administration were detected 30 min after dosing and persisted for up to 4 h. Administration of PCP via the s.c. route resulted in induction of more IEGs and consistently larger magnitudes of induction than that via the i.p. route. Therefore, we have outlined the dosing conditions to induce rapid and robust effect of acute PCP on behaviour, gene induction, and pharmacokinetic profile, to allow investigation of this as a potential animal model of acute psychosis.

Antipsychotic Treatment Alters Protein Expression Associated with Presynaptic Function and Nervous System Development in Rat Frontal Cortex

Haloperidol and olanzapine are widely used antipsychotic drugs in the treatment of schizophrenia and other psychotic disorders. Despite extensive research efforts within the biopharmaceutical industry and academia, the exact molecular mechanisms of their action remain largely unknown. Since the response of patients to existing medications can be variable and often includes severe side effects, it is critical to increase our knowledge on their mechanism of action to guide clinical usage and new drug development. In this study, we have employed the label-free liquid chromatography tandem mass spectrometry (LC-MSE) to identify differentially expressed proteins in rat frontal cortex following subchronic treatment with haloperidol or olanzapine. Subcellular fractionation was performed to increased proteomic coverage and provided insight into the subcellular location involved in the mechanism of drug action. LC-MSE profiling identified 531 and 741 annotated proteins in fractions I (cytoplasmic-) and II (membrane enriched-) in two drug treatments. Fifty-nine of these proteins were altered significantly by haloperidol treatment, 74 by olanzapine and 21 were common to both treatments. Pathway analysis revealed that both drugs altered similar classes of proteins associated with cellular assembly/organization, nervous system development/function (particularly presynaptic function) and neurological disorders, which indicate a common mechanism of action. The top affected canonical signaling pathways differed between the two treatments. The haloperidol data set showed a stronger association with Huntingtons disease signaling, while olanzapine treatment showed stronger effects on glycolysis/gluconeogenesis. This could either relate to a difference in clinical efficacy or side effect profile of the two compounds. The results were consistent with the findings reported previously by targeted studies, demonstrating the validity of this approach. However, we have also identified many novel proteins which have not been found previously to be associated with these drugs. Further study of these proteins could provide new insights into the etiology of the disease or the mechanism of antipsychotic medications.

Evaluation of the mGlu8 receptor as a putative therapeutic target in schizophrenia.

Aberrant glutamatergic neurotransmission may underlie the pathogenesis of schizophrenia and metabotropic glutamate receptors (mGluRs) have been implicated in the disease. We have established the localization of the group III mGluR subtype, mGluR8, in the human body and investigated the biological effects of the selective mGluR8 agonist (S)-3,4-dicarboxyphenylglycine ((S)-3,4-DCPG) in schizophrenia-related animal models. The mGlu8 receptor has a widespread CNS distribution with expression observed in key brain regions associated with schizophrenia pathogenesis including the hippocampus. (S)-3,4-DCPG inhibited synaptic transmission and increased paired-pulse facilitation in rat hippocampal slices supporting the role of mGluR8 as a presynaptic autoreceptor. Using the rat Maximal Electroshock Seizure Threshold (MEST) test, (S)-3,4-DCPG (30 mg/kg, i.p.) reduced seizure activity confirming the compound to be centrally active following systemic administration. (S)-3,4-DCPG did not reverse (locomotor) hyperactivity induced by acute administration of phenylcyclidine (PCP, 1-32 mg/kg, i.p.) or amphetamine (3-30 mg/kg, i.p.) in Sprague-Dawley rats. However, 10 nmol (i.c.v.) (S)-3.4-DCPG did reverse amphetamine-induced hyperactivity in mice although it also inhibited spontaneous locomotor activity at this dose. In addition, mGluR8 null mutant mouse behavioral phenotyping revealed an anxiety-related phenotype but no deficit in sensorimotor gating. These data provide a potential role for mGluR8 in anxiety and suggest that mGluR8 may not be a therapeutic target for schizophrenia.

Increased expression of the NR2A NMDA receptor subunit in the prefrontal cortex of rats reared in isolation

A hypofunction of the N‐methyl‐D‐aspartate (NMDA) receptor has been implicated in the pathophysiology of schizophrenia. Compelling evidence of altered NMDA receptor subunit expression in the schizophrenic brain has not, however, so far emerged. Rats reared in isolation exhibit several characteristics, including disturbed sensory gating, which resemble those seen in schizophrenia. To explore the possibility that NMDA receptor dysfunction may contribute to the behavioral and neurochemical consequences of rearing rats in isolation, we compared NMDA receptor subunit expression in brains of rats which were housed in isolation and which displayed a deficit in prepulse inhibition of the acoustic startle response with that of socially housed controls. An initial microarray analysis revealed a 1.26‐fold increase in NR2A transcript in the prefrontal cortex, but not in the nucleus accumbens, of rats reared in isolation compared with those housed socially. In contrast, NR1, NR2B, NR2C, NR2D, NR3A, and NR3B subunit expression was unchanged in either brain area. In a second cohort of animals, in situ hybridization revealed increased NR2A mRNA expression in the medial prefrontal cortex, an observation that was substantiated by increased [3H]CGP39653 binding suggesting that NR2A receptor subunit protein expression was also elevated in the medial prefrontal cortex of the same animals. No changes in expression of NR1 or NR2B subunits were observed at both mRNA and protein level. Altered NR2A subunit expression in the medial prefrontal cortex of rats reared in isolation suggests that NMDA receptor dysfunction may contribute to the underlying pathophysiology of this preclinical model of aspects of schizophrenia. Synapse 63:836–846, 2009.

Molecular cloning and characterisation of a novel GABAB-related G-protein coupled receptor

Using a homology-based bioinformatics approach we have analysed human genomic sequence and identified the human and rodent orthologues of a novel putative seven transmembrane G protein coupled receptor, termed GABA(BL). The amino acid sequence homology of these cDNAs compared to GABA(B1) and GABA(B2) led us to postulate that GABA(BL) was a putative novel GABA(B) receptor subunit. The C-terminal sequence of GABA(BL) contained a putative coiled-coil domain, di-leucine and several RXR(R) ER retention motifs, all of which have been shown to be critical in GABA(B) receptor subunit function. In addition, the distribution of GABA(BL) in the central nervous system was reminiscent of that of the other known GABA(B) subunits. However, we were unable to detect receptor function in response to any GABA(B) ligands when GABA(BL) was expressed in isolation or in the presence of either GABA(B1) or GABA(B2). Therefore, if GABA(BL) is indeed a GABA(B) receptor subunit, its partner is a potentially novel receptor subunit or chaperone protein which has yet to be identified.