Year

2015

Degree Name

Doctor of Philosophy

Department

School of Medicine

Abstract

Metabotropic glutamate receptor subtype 5 (mGluR5)-targeting therapeutics display a favourable profile of effects in preclinical paradigms, particularly for the treatment of cognitive dysfunction. It however remains unclear whether mGluR5 plays a causal or epiphenomenal role in the pathophysiology of schizophrenia. It is important to understand the aspects of mGluR5 that might be altered within the pathological condition, as this could have implications for novel drugs targeted at these systems in the patient. This thesis therefore aimed to deconstruct [at least some] of the complexities of mGluR5 in schizophrenia, with a focus on potential dysregulation of mGluR5, predominately utilising human tissues (postmortem brain tissues and DNA samples derived from schizophrenia patients).

Considering many symptoms associated with schizophrenia overlap with other closely related neuropsychiatric disorders, the first study in this thesis (Chapter 2) aimed to address whether mGluR5 alterations were specific to a particular set of symptoms (such as depressive symptoms or psychotic symptoms, or a combination thereof). To do this, mGluR5 binding was performed in postmortem samples from the anterior cingulate cortex, a region that modulates cognitive and emotional processes, of subjects with schizophrenia, major depression (with or without psychosis) or bipolar disorder. The results revealed that mGluR5 binding was unaltered across all neuropsychiatric pathologies, providing preliminary evidence that mGluR5 is not affected, at least in this region. Binding studies provide valuable insight into the ability of a receptor to interact with ligands and are thus useful for determining the therapeutic utility of a receptor; however this method is unable to detect wider changes, such as altered regulation, which may be underlying the observed pathologies.

Building on from this work, Chapter 3 aimed to explore whether dysregulation was present within the mGluR5 system in schizophrenia. Postmortem samples from the dorsolateral prefrontal cortex (DLPFC; BA46 involved in higher executive and cognitive processes) of schizophrenia subjects were examined. mGluR5, and proteins known to regulate mGluR5 trafficking, endocytosis and signalling (Norbin, Tamalin and Preso1) were examined. This study was also expanded to include measures of mGluR5 mRNA levels in the same subjects (data contributed by the S. Fung and C.S. Weickert). Together, this study revealed that although mGluR5 mRNA levels were unaltered, mGluR5 protein levels were greatly increased in this brain region. Conversely, mGluR5 endogenous regulators (Norbin, Tamalin and Preso1) were significantly decreased in this region. Interestingly, mGluR5 protein levels were correlated with levels of mGluR5 mRNA and the measured mGluR5 endogenous regulators in control subjects, but this correlation was lost in schizophrenia subjects. These results strongly support the presence of mGluR5 dysregulation in schizophrenia, at least in this region.

The work in Chapter 4 aimed to determine whether these findings in the DLPFC were exclusive to this brain region, or whether they extended to the hippocampus. The hippocampus is another region involved in schizophrenia pathology, which is involved in learning and memory functions. Extensive animal and cell-based data suggests mGluR5 critically mediates synaptic plasticity specifically in the CA1 subregion of the hippocampus. The results from this study thus found, that similarly to the DLPFC, mGluR5 was increased in the CA1 region. In contrast to the DLPFC, however, levels of mGluR5 endogenous regulators were significantly increased in this region, and no correlations between mGluR5 and the mGluR5 endogenous regulators were observed. These findings suggest that whilst alterations to mGluR5 extend to the CA1 region, the function, signalling, or regulation of mGluR5 might be brain-region specific. This work nonetheless also supports that mGluR5 is dysregulated in schizophrenia.

In Chapters 3 and 4, the influence of commonly used antipsychotic drugs (APDs) on mGluR5 was examined. In this series of studies, adult male rats were treated with either haloperidol (a first generation APD) or olanzapine (a second generation APD) for short, medium and long-term durations (APDS treated rat brains contributed by X.F. Huang). The protein levels of mGluR5 and mGluR5 endogenous regulators were subsequently measured in regions corresponding to the human studies, the PFC and hippocampus. The results indicated that commonly used APDs do not influence the mGluR5 system, suggesting that the postmortem findings in Chapters 2, 3 and 4 are not confounded by premortem mediation history. These findings also suggest that novel drugs to correct potential changes in mGluR5 might be useful as an adjunct treatment with current APDs, to target the observed alterations in the postmortem human tissues that are not targeted by current APDs.

The knowledge regarding mGluR5 is becoming increasingly substantial. Nonetheless, very little is known about the role of the GRM5 gene, encoding for mGluR5, in the genetic susceptibility to schizophrenia. Chapter 5 thus aimed to determine whether single nucleotide polymorphisms (SNPs) within the GRM5 gene are associated with schizophrenia diagnosis in a large Caucasian case-control population. In light of my previous works from Chapters 3 and 4, indicating mGluR5 dysregulation at the protein level, SNPs located in the 3’ untranslated region of GRM5 were selected, as this is a genomic region that is highly involved in protein conformation and regulation of regions responsible for protein-protein interactions. Furthermore, due to the extensive evidence that mGluR5 underlies cognitive processes, effects of genetic variability within the chosen SNPs were analysed for their effects on various measures of cognitive function (data contributed by M.J. Green). As hypothesised, the measured SNPs within GRM5 were differentially association with schizophrenia diagnosis. Further, genetic variability within GRM5 affected and/or was able to significantly predict the level of cognitive dysfunction in schizophrenia patients. Interestingly in this study, all findings were sex-specific, suggesting that GRM5/mGluR5 may be differently regulated in men and women with schizophrenia. Nonetheless, the association of SNPs located within the 3’ untranslated region of GRM5 contributes to the substantial evidence presented in this thesis that mGluR5 is dysregulated in the pathology of schizophrenia, and supports that mGluR5 has a modulatory effect on cognitive function in humans, not just animals.

Using a systematic approach to analyse mGluR5 from the gene-level to protein-level, and further, in human cognitive performance, the findings from this thesis provide the first compelling evidence that mGluR5 is dysregulated in schizophrenia. Although further studies are required to investigate the exact mechanisms responsible for this dysregulation, the findings presented here support the development of agents that modulate mGluR5 regulation and/or signalling to treat cognitive dysfunctions in patients with schizophrenia.

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