Investigation of G72 (DAOA) expression in the human brain

<p>Abstract</p> <p>Background</p> <p>Polymorphisms at the G72/G30 locus on chromosome 13q have been associated with schizophrenia or bipolar disorder in more than ten independent studies. Even though the genetic findings are very robust, the physiological role of the predicted G72 protein has thus far not been resolved. Initial reports suggested G72 as an activator of D-amino acid oxidase (DAO), supporting the glutamate dysfunction hypothesis of schizophrenia. However, these findings have subsequently not been reproduced and reports of endogenous human G72 mRNA and protein expression are extremely limited. In order to better understand the function of this putative schizophrenia susceptibility gene, we attempted to demonstrate G72 mRNA and protein expression in relevant human brain regions.</p> <p>Methods</p> <p>The expression of G72 mRNA was studied by northern blotting and semi-quantitative SYBR-Green and Taqman RT-PCR. Protein expression in human tissue lysates was investigated by western blotting using two custom-made specific anti-G72 peptide antibodies. An in-depth <it>in silico </it>analysis of the G72/G30 locus was performed in order to try and identify motifs or regulatory elements that provide insight to G72 mRNA expression and transcript stability.</p> <p>Results</p> <p>Despite using highly sensitive techniques, we failed to identify significant levels of G72 mRNA in a variety of human tissues (e.g. adult brain, amygdala, caudate nucleus, fetal brain, spinal cord and testis) human cell lines or schizophrenia/control post mortem BA10 samples. Furthermore, using western blotting in combination with sensitive detection methods, we were also unable to detect G72 protein in a number of human brain regions (including cerebellum and amygdala), spinal cord or testis. A detailed <it>in silico </it>analysis provides several lines of evidence that support the apparent low or absent expression of G72.</p> <p>Conclusion</p> <p>Our results suggest that native G72 protein is not normally present in the tissues that we analysed in this study. We also conclude that the lack of demonstrable G72 expression in relevant brain regions does not support a role for G72 in modulation of DAO activity and the pathology of schizophrenia via a DAO-mediated mechanism. <it>In silico </it>analysis suggests that G72 is not robustly expressed and that the transcript is potentially labile. Further studies are required to understand the significance of the G72/30 locus to schizophrenia.</p

Decreased striatal dopamine in group II metabotropic glutamate receptor (mGlu2/mGlu3) double knockout mice

Background: Group II metabotropic glutamate receptors (mGlu2 and mGlu3, encoded by Grm2 and Grm3) have been the focus of attention as treatment targets for a number of psychiatric conditions. Double knockout mice lacking mGlu2 and mGlu3 (mGlu2/3−/−) show a subtle behavioural phenotype, being hypoactive under basal conditions and in response to amphetamine, and with a spatial memory deficit that depends on the arousal properties of the task. The neurochemical correlates of this profile are unknown. Here, we measured tissue levels of dopamine, 5-HT, noradrenaline and their metabolites in the striatum and frontal cortex of mGlu2/3−/− double knockout mice, using high performance liquid chromatography. We also measured the same parameters in mGlu2−/− and mGlu3−/− single knockout mice. Results: mGlu2/3−/−mice had reduced dopamine levels in the striatum but not in frontal cortex, compared to wild-types. In a separate cohort we replicated this deficit and, using tissue punches, found it was more prominent in the nucleus accumbens than in dorsolateral striatum. Noradrenaline, 5-HT and their metabolites were not altered in the striatum of mGlu2/3−/− mice, although the noradrenaline metabolite MHPG was increased in the cortex. In mGlu2−/− and mGlu3−/− single knockout mice we found no difference in any monoamine or metabolite, in either brain region, compared to their wild-type littermates. Conclusions: Group II metabotropic glutamate receptors impact upon striatal dopamine. The effect may contribute to the behavioural phenotype of mGlu2/3−/− mice. The lack of dopaminergic alterations in mGlu2−/− and mGlu3−/− single knockout mice reveals a degree of redundancy between the two receptors. The findings support the possibility that interactions between mGlu2/3 and dopamine may be relevant to the pathophysiology and therapy of schizophrenia and other disorders.</p

Ionotropic Receptors in the Central Nervous System and Neurodegenerative Disease

Glutamate was identified as the main excitatory neurotransmitter in the mammalian central nervous system (CNS) following the observation in the early 1950s that glutamate can induce seizure activity and excite neurons in the mammalian brain. Over the last two decades, selective ligands, including competitive agonists and antagonists and allosteric modulators, have been developed to further investigate the functional role of glutaminergic receptors. Glutamate released from synapses can activate ligand-gated cation channels at postsynaptic cells to mediate fast postsynaptic potentials. These ion channel-forming ionotropic glutamate receptors (iGluRs) are divided into N-methyl-D-aspartate (NMDA), α-amino-3-hydroxyl-5-methyl-isoxazole-4-prorionate (AMPA), and kainate (KA) receptors. While only 20–30 % of the amino acid sequence is shared among these receptor subtypes, they share similar structural features and their activity is based on specific pharmacological preference. In this chapter, we will describe the structure and composition of iGluRs and infer their pharmacology, with a particular focus on their role in the CNS and their relevance to the pathogenesis of neurodegenerative diseases.22 page(s