Exploring group 1 metabotropic

glutamate receptors in the pathology

and treatment of schizophrenia

This thesis is presented as part of the requirement for the conferral

of the degree:

Doctor of Philosophy

Jeremy Lum, BMedHlthSc (Hons)

Supervisors:
Associate Professor Kelly Newell
Dr Lezanne Ooi
Distinguished Professor Xu-Feng Huang

School of Medicine
2018
 Declaration

I, Jeremy Lum, declare this thesis submitted in partial fulfilment of the requirements
for the conferral of the degree doctor of philosophy, from the University of
Wollongong, is wholly my own work unless otherwise referenced or acknowledged.
This document has not been submitted for qualifications at any other academic
institution.

Jeremy Lum

July, 2018
 Statements
In accordance with the University of Wollongong thesis committee ‘Guidelines for
Higher Degree Research Candidates on the Preparation and Submission of Higher
Degree Research Theses’ and ‘Higher Degree Research Thesis by Compilation Rules’
(2014) this PhD is presented as a ‘Thesis by Compilation’. It is comprised of a series of
four original studies published in or submitted to, peer-reviewed journals, of which I am
the first author. I hereby declare that I am a designer of these studies, have carried out
the experimental procedures (unless references or acknowledged), data analysis and
manuscript preparation.

Jeremy Lum

I consent to the presentation of this PhD in ‘Thesis by Compilation’ and I acknowledge

the above statement to be correct.

Associate Professor Kelly A Newell (Supervisor)

Jeremy Lum 3
 Publication and Presentations
The following publications and presentations have arisen directly from work contained
within this thesis.

Peer-Reviewed Publications
 Lum JS, Millard SJ, Frank E, Matosin N, Huang XF, Ooi L, Newell KA, “Chronic
adolescent CDPPB treatment alters short-term, but not long-term, glutamatergic
receptor expression”, In Press, Neurochemical Research. DOI: 10.1007/s11064-
018-2584-x
 Lum JS, Pan B, Deng C, Huang XF, Ooi L, Newell K, 2018. Effects of short- and
long-term aripiprazole treatment of group 1 mGluRs in the nucleus accumbens:
comparison with haloperidol. Psychiatry Research. 260, 152-157 DOI:
10.1016/j.psychres.2017.11.048.
 Lum JS, Millard SJ, Huang XF, Ooi L, Newell KA, 2017. A postmortem analysis of
NMDA ionotropic and group 1 metabotropic glutamate receptors in the nucleus
accumbens in schizophrenia. Journal of Psychiatry and Neuroscience. 42, 170077.
DOI: 10.1503/jpn.170077
 Lum JS, Fernandez, F., Matosin, N., Andrews, J.L., Huang, X-F., Ooi, L., Newell,
K.A., 2016. Neurodevelopmental Expression Profile of Dimeric and Monomeric
Group 1 mGluRs: Relevance to Schizophrenia Pathogenesis and Treatment.
Scientific Reports. 6, 34391. DOI: https://doi.org/10.1038/srep34391

Conference Presentations
 Lum J.S, Huang XF, Ooi L, Newell KA. The Neurodevelopmental Profile of
Dimeric and Monomeric mGluR5: Implications for Novel Therapeutics. ASCEPT-
MPGPCR Joint Scientific Meeting, Melbourne, November, 2016. (Poster)
 Lum JS, Frank E, Matosin N, Huang XF, Ooi L, Newell KA. Chronic Adolescent
CDPPB Treatment Causes Alterations to mGluR5 and Homer1b/c. Biological
Psychiatry Australia Conference, 2016, Newcastle, Australia, September, 2016.
(Poster)
 Lum J.S, Ooi L, Matosin N, Andrews JA, Huang XF, Fernandez F and Newell KA.
“The neurodevelopmental profile of group 1 metabotropic glutamate receptors:
Implications for novel therapeutics” International College of
Neuropsychopharmacology, South Korea, July, 2016. (Poster)
 Lum J.S, Ooi L, Matosin N, Fernandez F, Andrews JL, Huang XF, Newell KA,
Neurodevelopmental Expression of the Metabotropic Glutamate Receptor 5:

Jeremy Lum 4
 Relevance to the Perinatal PCP model of Schizophrenia, Society for Mental
Health Research, Brisbane, Australia, December, 2015. (Oral)
 Lum J.S, Ooi L, Matosin N, Huang XF, Newell KA. Evidence for dissociation of
mGluR5 and D2R in the nucleus accumbens in schizophrenia. Biological
Psychiatry Australia Conference, 2015, Coogee, Australia, 21-22 September,
2015. (Poster)

Community Presentations:
 Pregnancy, Pills and Progeny, Corrimal Rotary Club, Australian Rotary Health,
May, 2018,
 Investigating the Glutamatergic System in Animal Models of Psychiatric
Disorders, Illawarra Health and Medical Research Institute, Student Seminar
Series, September, 2016,
 Investigation a new Therapeutic Target for the Treatment of Schizophrenia,
Wollongong District Rotary Club, Australian Rotary Health, May, 2015,

Grants
 A.M Woods Consumables Grant, Schizophrenia Research Institute ($15 000)

Scholarships/Awards
 Mentor/Mentee Program, The International College of
Neuropsychopharmacology, 2016 ($500USD)
 Student Encouragement Award, The International College of
Neuropsychopharmacology, 2016 ($200USD)
 Ian Scott Scholarship (PhD top-up stipend), Australian Rotary Health, 2013-2017
($13 098)
 Fast-track Australian Postgraduate Award Scholarship. “Exploring group 1
metabotropic glutamate receptors in the pathology and treatment of
schizophrenia”, PhD stipend. Faculty of Science, Medicine and Health,
University of Wollongong, 2013-2017 ($75 000)
 Faculty Travel Grant, University of Wollongong ($2 000)
 Summer Research Scholarship, “Investigation of the antipsychotic, haloperidol,
on downstream signaling pathways in neurons derived from human induced
pluripotent stem cells”, Faculty of Health and Behavioural Science, University of
Wollongong, 2013 ($2 000)

Jeremy Lum 5
 Additional Publications and Presentations
The following publications and presentations have arisen as a result of other projects I
have been involved in during my doctoral studies:
Peer-Reviewed Publications
 Balez R, Steiner N, Engel M, Sanz Munoz S, Lum JS, Wu Y, Wang D, Vallotton P,
Sachdev P, O'Connor M, Sidhu K, Münch G and Ooi L, Neuroprotective effects of
apigenin against inflammation, neuronal excitability and apoptosis in an induced
pluripotent stem cell model of Alzheimer's disease" Scientific Reports. 2016
 Matosin N, Fernandez-Enright F, Lum JS, Engel M, Andrews JL, Gassen NC,
Wagner KV, Schmidt MV, Newell KA. Molecular evidence of synaptic pathology
in CA1 in schizophrenia. npj Schizophrenia. May 2016.
 Matosin N, Fernandez-Enright F, Lum JS, Newell KA. Shifting towards a model of
mGluR5 dysregulation in schizophrenia: consequences for future schizophrenia
treatment. Invited review, special Issue: “mGluRs: 5 years on”.
Neuropharmacology. 2015.
 Matosin N, Fernandez-Enright F, Lum JS, Andrews JL, Engel M, Huang XF, Newell
KA. Metabotropic glutamate receptor 5 and its trafficking molecules, Norbin and
Tamalin, are increased in the CA1 hippocampal region of subjects with
schizophrenia. Schizophrenia Research. 2015. 166(1-3): 212-218.
 Matosin N, Fernandez-Enright F, Fung SJ, Lum JS, Engel M, Andrews JL, Huang XF,
Weickert CS, Newell KA. Alterations of mGluR5 and its endogenous regulators
Norbin, Tamalin and Preso1 in schizophrenia: towards a model of mGluR5
dysregulation. Acta Neuropathologica. 2015. 130(1): 119-129.
 Matosin N, Frank E, Engel M, Lum JS, Newell KA. Negativity towards negative
results: a discussion of the disconnect between scientific worth and scientific
culture (editorial). Disease Models & Mechanisms. 2014. 7(2): 171-173.

Book chapter
 Newell KA, Matosin N, Lum JS. Metabotropic glutamate receptors in the
pathophsyiology and treatment of schizophrenia and major depression (book
chapter). Metabotropic Glutamate Receptors: Molecular Mechanisms, Role in
Neurological Disorders and Pharmacological Effects. Nova Publishers, 2014.

Jeremy Lum 6
Conference Presentations
 Lum JS, Bird KM, Wilkie J, Millard SJ, Pallimulla S, Newell KA, Wright IM,
“Maternal methadone treatment causes cognitive deficits in adolescent
offspring behaviour: A rodent study”, 32nd Annual Fetal and Neonatal Workshop
of Australia and New Zealand, Queenstown, New Zealand, 2018. (Oral)
 Lum JS, Reminis NS, Eiby YA, Lingwood BE, Wright IMR, “Stinky babies: A role for
H2S in the cardiac instability of preterms”, 44th Annual Meeting Fetal and
Neonatal Physiology Society, Osaka, Japan, 2017 (Oral)
 Lum JS, Mackay C, Huang XF, Fernandez F, Newell KA, “Glutamatergic alterations
in the Hippocampus of a Rodent Depression Model”, Kioloa Neuroscience
Colloquium, Kioloa NSW, Australia, 2016. (Oral)
 Lum JS, Mackay C, Millard S, Huang XF, Fernandez, Newell KA. The Emergence
of Behavioural and Neurochemical Changes at Adolescence in a Rodent Model
of Depression. Australian Society of Medical Research, Sydney, 2016 (Poster)
 Lum JS, Mackay C, Millard S, Huang XF, Fernandez, Newell KA. The Emergence
of Behavioural and Neurochemical Changes at Adolescence in a Rodent Model
of Depression. Biological Psychiatry Australia Conference, 2016, Newcastle,
Australia, September 2016. (Poster)

Conference Abstracts
 Newell KA, Mackay C, Lum JS, Millard S. Huang XF and Fernandez F. Norbin: an
emerging player in the pathophysiology and treatment of depression.
International Journal of Neuropsychopharmacology, Volume 19, Issue Suppl_1,
1 June 2016, Pages 50, https://doi.org/10.1093/ijnp/pyw043.146 (Poster)
 Newell KA, Millard S, Mackay C, Lum JS, Ramos M, Webby R, and Fernandez F.
Maternal fluoxetine treatment influences anxiety- and depressive-like
behaviours in adolescent offspring: a rodent model. Int J
Neuropsychopharmacol. 2016 Jun; 19(Suppl 1): 44 10.1093/ijnp/pyw043.130
(Poster)
 Millard S, Fernandez F, Mackay C, Lum JS, Ramos M, Webby R, Newell KA.
Maternal Fluoxetine (Prozac) treatment influences anxiety-like and depressive-
like behaviour in exposed offspring”, Kioloa Neuroscience Colloquium, Kioloa
NSW, Australia, 2016 (Poster)
 Matosin N, Engel M, Fernandez-Enright F, Lum JS, Andrews JL, Huang XF, Newell
KA. Alterations of mGluR5 and mGluR5 signaling partners in schizophrenia.
Society for Neuroscience Meeting, Washington DC 15-19 November 2014.
(Poster)

Jeremy Lum 7
  Matosin N, Green MJ, Andrews JL, Lum JS, Engel M, Huang XF, Fernandez-Enright
F* and Newell KA*. mGluR5 dysregulation in schizophrenia and cognition: from
gene to protein. Society of Biological Psychiatry 70th Anuual Meeting, Toronto
Canada, May 14-16 2015. Late breaking poster. *equal contribution (Poster)
 Matosin N, Green MJ, Andrews JL, Lum JS, Engel M, Huang XF, Fernandez-Enright
F and Newell KA. mGluR5 dysregulation in schizophrenia and cognition: from
gene to protein. 25th ISN-APSN Biennial Meeting in conjunction with ANS, Cairns,
Australia August 23-27, 2015. *equal contribution (Poster)

Grants:
 Daniel Beck Memorial Award for Schizophrenia Research, NeuRA ($2 000)

Awards:
 Best Oral Presentation-Early Investigator, 32nd Annual Fetal and Neonatal
Workshop of Australia and New Zealand, Queenstown, New Zealand, 2018.
($400)

Jeremy Lum 8
 Tables of Contents

Declaration .................................................................................................................................... 2
Statements .................................................................................................................................... 3
Publication and Presentations ...................................................................................................... 4
Additional Publications and Presentations ................................................................................... 6
Tables of Contents ........................................................................................................................ 9
List of Figures .............................................................................................................................. 11
List of Tables ............................................................................................................................... 12
List of Abbreviations ................................................................................................................... 13
Abstract ....................................................................................................................................... 15
Acknowledgments....................................................................................................................... 18
Chapter 1..................................................................................................................................... 20
1.1 Schizophrenia .................................................................................................................... 20
1.1.1 The Pathogenesis and Neurodevelopment Hypothesis of Schizophrenia ................. 20
1.1.2 The Neurobiology of Schizophrenia ........................................................................... 21
1.2 Glutamate receptors ......................................................................................................... 24
1.3 The Hypoglutamatergic Hypothesis of Schizophrenia ...................................................... 26
1.4 Group 1 mGluRs ................................................................................................................ 32
1.4.1 Expression and Splice Variants................................................................................... 32
1.4.2 Signalling .................................................................................................................... 32
1.4.3 Neurodevelopmental Profile...................................................................................... 35
1.4.4 Structure .................................................................................................................... 36
1.5 Group 1 mGluR Interacting Partners-Homer1 and Norbin ............................................... 42
1.5.1 Homer1 ...................................................................................................................... 42
1.5.2 Norbin/Neurochrondrin ............................................................................................. 43
1.6 Group 1 mGluRs and their endogenous regulators in the pathology of schizophrenia ... 44
1.6.1 Group 1 mGluRs-Evidence from knock-out and pharmacological models ................ 44
1.6.2 Group 1 mGluRs-Evidence from postmortem studies ............................................... 46
1.6.3 Homer1 ...................................................................................................................... 50
1.6.4 Norbin ........................................................................................................................ 51
1.7 Effect of Current Antipsychotic Drugs on Group 1 mGluR................................................ 52
1.8 mGluR5 positive allosteric modulation as a therapeutic target for schizophrenia .......... 57
1.8.1 Therapeutic potential of CDPPB ................................................................................ 59
Jeremy Lum 9
 1.9 Summary ........................................................................................................................... 66
1.10 Aims and Hypotheses...................................................................................................... 68
1.10.1 General Aim: ............................................................................................................ 68
1.10.2 Specific Aims: ........................................................................................................... 68
1.2.3 Rationale and Hypotheses ......................................................................................... 68
1.11 Significance ..................................................................................................................... 70
Chapter Two ................................................................................................................................ 71
2.1 Study rationale and aims .................................................................................................. 71
2.2 Manuscript details ............................................................................................................ 71
2.3 Author contributions......................................................................................................... 71
2.4 Collaborator’s statement .................................................................................................. 72
Chapter Three ............................................................................................................................. 91
3.1 Study rationale and aims .................................................................................................. 91
3.2 Manuscript details ............................................................................................................ 91
3.3 Author contributions......................................................................................................... 91
3.4 Collaborator’s statement .................................................................................................. 92
Chapter Four ............................................................................................................................... 99
4.1 Study rationale and aims .................................................................................................. 99
4.2 Manuscript details .......................................................................................................... 100
4.3 Author contributions....................................................................................................... 100
4.4 Collaborator’s statement ................................................................................................ 100
Chapter Five .............................................................................................................................. 112
5.1 Study rationale and aims ................................................................................................ 112
5.2 Manuscript details .......................................................................................................... 112
5.3 Author contributions....................................................................................................... 112
5.4 Collaborator’s statement ................................................................................................ 113
Chapter Six ................................................................................................................................ 123
6.1 Overall discussion ........................................................................................................... 123
6.2 Limitations....................................................................................................................... 137
6.3 Future Directions ............................................................................................................ 139
6.4 Conclusion ....................................................................................................................... 143
References ................................................................................................................................ 144

Jeremy Lum 10
 List of Figures
Figure 1.1: Illustration of G-protein-dependent and independent potentiation of the NMDA
receptor ...................................................................................................................................... 34
Figure 1.2: Schematic of proposed activation of dimeric and monomeric mGluRs by orthosteric
agonists and positive allosteric modulators ............................................................................... 40
Figure 6.1: Immunoblots of rodent cortical tissue with mGluR1 and mGluR5 ........................ 131
Figure 6.2: Representative immunoblot images of mGluR5 in the frontal cortex of Sprague-
Dawley and Wistar-Kyoto rats at postnatal days 12, 35 and 96.. ............................................. 133

Jeremy Lum 11
 List of Tables
Table 1.1: Summary of studies investigating the neurodevelopmental mRNA and protein
expression profile of mGluR1 and mGluR5. .................................................................................37
Table 1.2: Summary of mGluR1 and mGluR5 investigations in postmortem human brain tissue
from schizophrenia patients. .......................................................................................................48
Table 1.3: Summary of preclinical studies assessing the behavioural and neurochemical efficacy
of CDPPB in rodents. ....................................................................................................................63

Jeremy Lum 12
 List of Abbreviations
[3H] Tritiated
AAV Adeno-associated virus
Akt Protein kinase B
AMPA α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
BA Brodmann area
BDNF Brain-derived neurotropic factor
CA Cornu Ammonis
CaM Calmodulin
CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide)
CNS Central nervous system
C-terminusCarboxyl terminus
CREB cAMP response element binding
DAG Diacylglycerol
DHPG (S)-3,5-Dihydroxyphenylglycine
EVH1 Enabled/VASP homology 1 domain
GABA γ-Aminobutyric acid
GKAP Guanylate-kinase-associated protein
GPCR G-protein coupled receptor
GRK G-protein regulatory kinase
IP3 Intracellular inositol 1,4,5-triphosphate
KO Knock-out
LTD Long-term depression
LTP Long-term potentiation
mGluR Metabotropic glutamate receptor
MK-801 [5R,10S]-[+]-5-,ethyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-
imine
MPEP 2-Methyl-6-(phenylethynyl)pyridine
mTOR Mammalian target of rapomycin
NMDA N-methyl-D-aspartate
PAM Positive allosteric modulator
PCP Phencyclidine
PET Positron emission tomography
PKC Protein kinase C
PLC Phospholipase C
PN Postnatal day
PSD Post-synaptic density
PSD-95 Post-synaptic density 95
Pyk2/CAKβ Proline-rich tyrosine kinase/cell adhesion kinase β
Jeremy Lum 13
SHANK SH3 and multiple ankyrin repeated domains
WT Wild-type

Jeremy Lum 14
 Abstract

There is a growing body of evidence indicating developmental disturbances to the

glutamatergic system may underlie the pathogenesis of schizophrenia. Genetic and
environmental studies indicate schizophrenia is associated with reduced glutamatergic
signalling, in particular via the primary ionotropic N-methyl-D-aspartate (NMDA)
glutamate receptor. Therefore, upregulation of NMDA receptor activity is proposed to
be a novel avenue for the treatment of schizophrenia. Group 1 metabotropic glutamate
receptors (mGluR), which include mGluR1 and mGluR5, are homodimeric G-protein
coupled receptors that co-localise with and in-directly potentiate NMDA receptor
currents. Therefore, group 1 mGluRs have been implicated in the pathology of
schizophrenia and identified as a novel treatment target. The aim of this thesis was to
assess group 1 mGluRs, particularly the dimeric and monomeric forms, in the
neurodevelopment, pathology and treatment of schizophrenia. In addition, this thesis
aimed to identify the neurochemical changes induced by treatment with the mGluR5
positive allosteric modulator, CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-
yl)benzamide), in an attempt to better understand its potential preclinical antipsychotic
efficacy.

Building on recent studies from our lab identifying altered group I mGluRs in the
prefrontal cortex and hippocampus in schizophrenia, Chapter 2 aimed to explore
potential glutamatergic deficits in the nucleus accumbens in schizophrenia, a region
believed to be central to schizophrenia pathology and receives glutamatergic
projections from the prefrontal cortex and hippocampus. Using the largest nucleus
accumbens postmortem cohort to date (n=30 control/30 schizophrenia), we report that
protein expression of dimeric and monomeric mGluR1 and mGluR5 as well as their
endogenous regulators, Norbin and Homer1b/c, in addition to the NMDA receptor
subunits, NR1, NR2A and NR2B were not altered in schizophrenia. Collectively, these
findings suggest the proteins may be altered in a brain-region specific manner,

Jeremy Lum 15
particularly regions associated with higher order cognitive functions, such as the
prefrontal cortex and hippocampus.

Chapter 3 aimed to investigate the protein expression of group 1 mGluRs, Norbin,

Homer1a and Homer1b/c in the nucleus accumbens following antipsychotic treatment,
using an established and clinically relevant rodent model. Adult rats were orally
administered daily doses of the typical antipsychotic, haloperidol (0.3 mg/kg), the
atypical antipsychotic, aripiprazole (2.25 mg/kg) or vehicle for 1- or 10-weeks. Although
group 1 mGluR expression was not altered following antipsychotic treatment, 1-week
treatment with haloperidol and aripiprazole increased Norbin and Homer1a protein
levels, respectively. Furthermore, Norbin protein expression was increased following 10-
week aripiprazole treatment. The observed differential temporal changes in Norbin and
Homer1a protein expression following antipsychotic treatment, may influence group 1
mGluR localisation and signalling which may partially mediate the therapeutic effects of
haloperidol and aripiprazole.

Chapter 4 aimed to investigate the neurodevelopmental expression of dimeric and

monomeric group 1 mGluRs, at critical neurodevelopmental time-points (postnatal day
(PN) 12, 35 and 96) in the perinatal phencyclidine (PCP) model of schizophrenia, to
provide insight into their possible role in the development of schizophrenia, but also
gain perspective into potential treatment windows for targeting mGluR5. We report in
the prefrontal cortex, hippocampus and nucleus accumbens, dimeric mGluR5 remained
relatively stable throughout PN12-PN96, however monomeric mGluR5 was abundantly
expressed at PN12 and substantially declined at PN36 and PN96. In contrast, mGluR1
monomer was consistently expressed throughout the time-points. Perinatal PCP
treatment increased dimeric mGluR5 levels in the frontal cortex and hippocampus,
however decreased hippocampal mGluR1 dimer levels at PN12. The early alterations
observed to group 1 mGluR expression in this model, may contribute to the
neurodevelopmental disruptions observed in this model and schizophrenia
pathogenesis. Furthermore, these findings indicate group 1 mGluRs exhibit unique

Jeremy Lum 16
dimeric and monomeric neurodevelopmental expression patterns, which may have
functional implications for their signalling and pharmacology.

There has been recent focus on group 1 mGluRs as therapeutic targets for treating
schizophrenia. In particular, mGluR5 PAMs such as CDPPB, have shown encouraging
efficacy in attenuating schizophrenia-like behaviours analogous to all symptom
domains, in rodent models. Recent studies examining the efficacy of CDPPB indicate
administration at adolescence may attenuate the development of schizophrenia-like
behaviours at adulthood, particularly cognitive function. Chapter 5 aimed to examine
the short- and long-term neurochemical effects following adolescent CDPPB treatment.
Immunoblot analysis of the adolescent hippocampus revealed CDPPB treatment
increased mGluR5, NMDA receptor subunit (NR1 and NR2A) and AMPA receptor subunit
(GluA1 and GluA2) expression. However, these changes were not observed at adulthood
or in the frontal cortex. These findings suggest adolescent CDPPB treatment may induce
brain-region specific effects on glutamatergic tone, which do not extend to adulthood.
Further understanding the neurochemical changes caused by mGluR5 positive allosteric
modulation, will improve drug design, ensuring their clinical suitability and efficacy.

Collectively, this thesis adds to the current body of research investigating the role of
group 1 mGluRs in the pathology, current and potentially future treatment of
schizophrenia. This work builds from the human neurobiology and progresses towards
neurodevelopmental, neuropharmacological, ultimately culminating in a novel
treatment strategy for schizophrenia, using a rodent model. Unravelling the
neurochemical changes that do or do not characterise schizophrenia and those
important for therapeutic efficacy, are essential in developing our understanding of
schizophrenia pathology and will consequently improve treatment approaches.

Jeremy Lum 17
 Acknowledgments

Several individuals have contributed to this thesis, in addition to my personal

development, for which I would like to extend my sincere gratitude for their support.
First and foremost, my primary supervisor Associate Professor Kelly Newell, who has
provided me with continual encouragement, support and opportunities throughout the
time I have spent in her research group. I appreciate your commitment to my progress,
the importance you have placed on helping achieve my goals and the copious amount
of time you have invested discussing scientific ideas, research approaches, life and
everything in between. Your advice has been invaluable and consequently shaped me
to be a better scientist and person.

To my co-supervisory team, Dr Lezanne Ooi and Distinguished Professor Xu-Feng Huang,

from the beginning you have provided me with your full support. Lezanne, your scientific
knowledge and commitment to research, never ceases to amaze me. The input and
encouragement you have provided, has been a tremendous support. Xu-Feng, thank you
for your generosity and willingness to share your resources, knowledge and experience.
Thank you for always making me feel a valued member of your team and ensuring we
work in a positive team environment. In addition, thank you to Professor Ian Wright for
your support to help me cross the finish line. Thank you all, for including me within your
talented research teams, who have been a massive support network, in particular,
Monique, Rachelle and Sonia.

To those who I shared the lab and office with, in particular Sam, Ash, Natalie, Martin,
Claire, Luke, BJ, Hongqin and Lisa, thank you for all your help within the lab, animal
house, scientific discussions and motivational talks. Thank you for sharing your technical
expertise and always offering a helping hand. To my best friends, Nat and Dzung, the
morning coffee dates have not only provided me with a reason to get up so early in the
morning, but also provided a place to bask in your caring, humorous and loving nature.

The present work was supported by the Illawarra Health and Medical Research Institute
(IHMRI), which provided the infrastructure and facilities to undertake this research.

Jeremy Lum 18
Thank you to IHMRI’s technical staff, for all your day-to-day help and constant drums of
TBST. I would also like to thank the Schizophrenia Research Institute and Australian
Rotary Health who generously provided financial support. Furthermore, Australian
Rotary Health for providing numerous professional development training sessions and
the opportunity to present our research to the public.

Finally, I could not have achieved anything without the lifetime of love and support from
my grandparents, aunties, uncles, cousins, brothers and sister, but particularly Mum,
Dad and Marleigh. You have sacrificed so much to provide me with everything I could
have asked for. Thank you for always being a foundation of support and a continual
reminder of what I can aspire to.

Jeremy Lum 19
 Chapter 1

1.1 Schizophrenia
Schizophrenia is a complex psychiatric disorder, which causes a lifetime affliction for
affected individuals. The prevalence of schizophrenia is approximately 0.7% of the
world’s population, with onset of symptoms occurring in early adulthood of males and
slightly later in females (Saha et al., 2005). Schizophrenia is diagnosed according to the
Diagnostic and Statistical Manual of Mental Disorders and is centred on the profile of an
individual’s symptoms. Symptoms of schizophrenia include positive (e.g. hallucinations
and delusions) and negative (e.g. depression, anxiety, social withdrawal and anhedonia),
with many patients also experiencing cognitive deficits (e.g. memory and learning
deficits) (Lewis and Lieberman, 2000). Cognitive impairments associated with
schizophrenia have shown to contribute to poor functional outcomes and are now
considered core to schizophrenia pathology (Green, 2006). Whilst antipsychotic drugs
are primarily used to treat the positive symptoms associated with schizophrenia, the
negative and cognitive symptoms are not adequately addressed by current treatments
(Buchanan et al., 2005; Kirkpatrick et al., 2006). Therefore, it is vital to develop an
understanding of schizophrenia to enable the advancement of more effective
treatments, particularly for associated cognitive impairments.

1.1.1 The Pathogenesis and Neurodevelopment Hypothesis of Schizophrenia

Despite the classification of schizophrenia over 100 years ago, its cause remains elusive.
It has been postulated that the genesis of schizophrenia begins before the onset of
classical symptoms, giving rise to the neurodevelopmental hypothesis of schizophrenia.
The foundations of the neurodevelopmental hypothesis propose that disruptions during
early development may underlie the pathogenesis of schizophrenia (McGrath et al.,
2003). In support of this hypothesis, factors during fetal development such as hypoxia,
maternal stress, malnutrition and maternal infection may cause increased risk of
developing schizophrenia in later life (Brown, 2006; Brown and Susser, 2008; Cannon et
al., 2002; Khashan et al., 2008). Reports from epidemiological studies show increased

Jeremy Lum 20
risk of schizophrenia in individuals exposed to famine or influenza in utero (Brown and
Susser, 2002). Furthermore, those born in winter or suffered birth complications have
higher incidence of diagnosis, suggesting the influence of early environmental factors
may increase the susceptibility of an individual to develop schizophrenia (Bradbury and
Miller, 1985). In addition, retrospective studies indicate a schizophrenia prodrome
characterised by delayed motor development, language difficulties and lower IQ
(Schenkel and Silverstein, 2004; Woodberry et al., 2008). Collectively, this evidence
supports a neurodevelopmental cause for the development of schizophrenia.

Despite a known definitive cause, there is strong evidence an interaction of genetic and
environmental factors contributes to the development of schizophrenia. In particular,
genes involved in brain development and synaptic plasticity, such as neuregulin 1
(NRG1) (Bakker et al., 2004), dysbindin (DTNBP1) (Schwab et al., 2003) and disrupted in
schizophrenia 1 (DISC1) (Callicott et al., 2005) have been associated with schizophrenia.
Whilst there are several risk alleles associated with schizophrenia, these genes are also
often associated with other psychiatric disorders such as bipolar, attention deficit
hyperactive disorder, autism and depression (Kilpinen et al., 2008; Wen et al., 2016). No
single gene has been identified to be responsible for schizophrenia, and not all
individuals exhibiting genetic traits are diagnosed with schizophrenia, suggesting the
impact of additional factors. Whilst there are several candidate genes and known
lifestyle factors, it is highly likely a combination of influences contribute to disrupted
neurodevelopmental processes and consequently to the pathogenesis of schizophrenia.

1.1.2 The Neurobiology of Schizophrenia

The exact cause(s) of schizophrenia are not known and there remains no diagnostic
marker or clear neuropathology, likely due to the heterogeneity of the disorder.
However, one of the most consistent findings comes from in vivo imaging studies, where
schizophrenia patients exhibit increased ventricular volume, accompanied by reduced
temporal lobe volume (Wright et al., 2000). Postmortem studies consistently report
reduced cortical inhibitory interneurons suggesting alterations in brain neurochemistry,
particularly those systems that regulate excitatory and inhibitory tone (Lewis et al.,

Jeremy Lum 21
2012). Furthermore, it is apparent from the multitude of animal and human studies that
the symptoms of schizophrenia are associated with alterations in neurotransmission.

While several neurotransmitter systems are implicated in the pathogenesis of

schizophrenia, it is widely accepted that altered subcortical dopamine is one important
component (Howes and Kapur, 2009). Several studies have reported increased levels of
the dopamine precursor, L-3,4-dihydroxyphenylalanine, in the striatum of schizophrenia
patients, indicating increased dopamine production and storage in presynaptic vesicles
(Meyer-Lindenberg et al., 2002; McGowan et al., 2004; Hietala et al., 1995, 1999; Howes
et al., 2009; Lindström et al., 1999; Reith et al., 1994). Furthermore, all studies that have
examined patients at a period of acute psychosis, observed elevated striatal presynaptic
dopamine levels, signifying increased dopamine production may be involved in the
pathogenesis of psychosis in schizophrenia (Hietala et al., 1995, 1999; Howes et al.,
2009; Lindström et al., 1999).

Following evidence of increased dopamine production, studies have examined the

release of dopamine using positron emission tomography (PET) and single photon
emission computerised tomography imaging. These studies rely on the competitive
binding of radiolabelled ligands to displace endogenous dopamine from dopamine
receptors to in-directly measure extracellular dopamine concentrations (Laruelle, 2000).
Several studies have investigated the release of dopamine in the striatum of
schizophrenia and control patients prior to and following amphetamine administration.
All studies reported no differences in baseline dopamine release between groups,
however following amphetamine administration, schizophrenia patients exhibit
significantly higher amounts of dopamine release (Laruelle et al., 1996; Breier et al.,
1997; Abi-Dargham et al., 1998). Furthermore, administration of amphetamine, which
increases synaptic dopamine, has been demonstrated to exacerbate positive symptoms
in several schizophrenia patients (Breier et al., 1997). These reports of increased
sensitivity to amphetamine challenges support the hypothesis that patients are at
increased susceptibility to dopamine sensitivity and further implicates subcortical
dopamine imbalance in schizophrenia pathology.

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In line with the evidence of dopaminergic abnormalities observed in schizophrenia,
blockade of subcortical hyperdopaminergic states, predominantly via antagonism of the
D2 receptor subtype, is the primary mechanism proposed for the therapeutic efficacy of
many current antipsychotic drugs (Grace et al., 1997). All current antipsychotics have
affinity for the D2 receptor, which is strongly correlated to their therapeutic ability to
reduce positive symptoms (Agid et al., 2006). Furthermore, current antipsychotics are
thought to exert this effect via the ventral striatum, in particular the nucleus accumbens,
making it a region of interest in the pathology and treatment of schizophrenia (Deutch
et al., 1992; Deutch and Cameron, 1992).

Although there is strong evidence for subcortical hyperdopaminergia, it cannot account

entirely for the pathology of schizophrenia for several reasons. Firstly, D2 receptor
targeting antipsychotics do not treat all symptoms. Current antipsychotics are primarily
effective for treating positive symptoms, however they largely have little effect on
negative and cognitive symptoms (Buchanan et al., 2005; Kirkpatrick et al., 2006).
Furthermore, not all patients respond to current antipsychotics (termed treatment
refractory schizophrenia) (Lindenmayer, 2000), which suggests other neurotransmitter
systems are likely involved. In line with this, a recent imaging study examining a small
cohort of schizophrenia patients (6-10/per group), reported those who responded to
dopaminergic antipsychotics displayed elevated striatal dopamine synthesis, whilst non-
responding patients displayed dopamine levels similar to controls (Demjaha et al.,
2014). Furthermore, non-responding patients displayed elevated glutamatergic
abnormalities in the anterior cingulate cortex, suggesting a subset of patients may
possess a different underlying pathophysiology and may require a different avenue of
treatment, possibly via targeting the glutamatergic system. Imaging studies, which have
been able to differentiate sub-regional differences of dopamine metabolism and
receptor binding, indicate that the nucleus accumbens may not show as exaggerated
dopaminergic abnormalities as previously thought (Howes et al., 2009; Kegeles et al.,
2010). In line with this, recent postmortem studies examining the nucleus accumbens
indicate that the rate limiting dopamine synthesis enzyme, tyrosine hydroxylase, is
unchanged (McCollum et al., 2016; McCollum and Roberts, 2015). Furthermore, protein

Jeremy Lum 23
expression of the vesicle glutamate transporter, VGLUT2 in the nucleus accumbens was
reported to be elevated in schizophrenia subjects (McCollum and Roberts, 2015). In
addition, increased asymmetric synapses, representative of glutamatergic projections
have been observed within the core of the nucleus accumbens in schizophrenia,
however these synapses showed reduced post-synaptic area (McCollum et al., 2015).
Collectively, these studies provide evidence for the presence of glutamatergic
abnormalities in the nucleus accumbens. Whilst this evidence is relatively recent, a
plethora of earlier evidence has implicated the glutamatergic system in the
pathophysiology of schizophrenia.

1.2 Glutamate receptors

The amino acid, glutamate, acts as the primary excitatory neurotransmitter in the
central nervous system (CNS). Glutamate plays an integral role in a wide variety of
neurodevelopmental processes including neurogenesis, neuronal migration and
synaptogenesis. Furthermore, glutamate is involved in mature neuronal functions such
as synaptic plasticity and neuronal signalling, processes that are disturbed in a range of
disorders including schizophrenia (McDonald and Johnston, 1990; Pearce et al., 1987).

Glutamate acts via two distinct receptor classes, ionotropic and metabotropic.
Ionotropic receptors include the N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-
methylisoxazole-4-proprionic acid (AMPA) and kainate receptors (Goff and Wine, 1997).
Activation of these receptors by glutamate causes a fast influx of Ca2+ or Na+ ions. The
expression and activity of ionotropic glutamate receptors, in particular the NMDA
receptor (discussed later in Section 1.3), has been vastly implicated in
neurodevelopmental psychiatric disorders such as schizophrenia. The NMDA receptor is
an assembly of heteromeric subunits, comprising at least one requisite NR1 subunit, in
complex with NR2 and/or NR3 subunits, creating a variety of different NMDA receptor
arrangements (Paoletti et al., 2013). The distinct composition and formation of these
subunits can produce a diversity of functional and pharmacological characteristics (Flint
et al., 1997; Kristiansen et al., 2007; Sheng et al., 1994).

Jeremy Lum 24
Although the NR1 subunit is obligatory for the NMDA receptor, it does not contain the
glutamate binding site; rather it possesses a binding site for the co-activator, glycine. It
is the NR2 subunits that contain the glutamate-binding site to act as the primary
activator and regulator of NMDA receptor function. There are four genes that encode
the different NR2 subunits (GRIN2A-D), which exhibit different spatiotemporal
regulation. The NR2C subunits are predominantly confined to the cerebellum and the
NR2D subunits to the thalamus and brainstem (Watanabe et al., 1992; Wenzel et al.,
1997). However, the NR2A and NR2B subunits are abundantly expressed in the cortex,
hippocampus and striatum, regions implicated in schizophrenia, making them of
particular interest for schizophrenia research (Portera-Cailliau et al., 1996). In regards to
the temporal expression of the NR2 subunits, rodent studies demonstrate NR2B and
NR2D are the predominant subunits expressed in the embryonic brain (Monyer et al.,
1994; Ritter et al., 2002; Wenzel et al., 1997). NR2A mRNA and protein expression
appear in the first few days following birth and steadily increase until it peaks at around
three postnatal weeks of age. Similarly, NR2C expression does not appear until after the
first week of postnatal development (Guilarte and McGlothan, 1998; Zhong et al., 1995).
In contrast, NR2B mRNA and protein expression increases following birth, however
plateaus within the first week of postnatal development, with high expression in
forebrain regions (Portera-Cailliau et al., 1996; Sheng et al., 1994; Zhong et al., 1995).

Similar to rodent studies, mRNA and protein examination of the human foetal brain has
shown NR2B is the primary NR2 subunit expressed in the cortex. Cortical NR2B
expression has been detected as early as eight weeks gestational age and progressively
increases during gestation (Ritter et al., 2001). Within the hippocampus, NR2B mRNA
expression reaches peak expression in the neonatal brain and slowly declines
throughout development into adulthood (Law et al., 2003). In contrast, NR2A expression
is low during gestation, however increases in expression following birth and remains
constant throughout life (Law et al., 2003; Ritter et al., 2001).

Glutamate also signals via the metabotropic glutamate receptors (mGluR). mGluRs are
seven transmembrane domain proteins and belong to the Class C G-protein coupled

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receptor family. mGluRs are sub-classified into three groups (I, II, III) based on their
sequence homology, G-protein coupling and pharmacology. Group I mGluRs comprise
of mGluR1 and mGluR5 and are located primarily on the post-synaptic membrane. They
are positively coupled to Gq/G11, instigating the activation of phospholipase C (PLC) and
a pathway of downstream responses, resulting in neuronal excitation. Group II mGluRs
include mGluR2 and mGluR3, whereas mGluR4, mGluR6, mGluR7 and mGluR8
encompass group III. Group II and III mGluRs are both negatively linked to adenylyl
cyclase and generally expressed pre-synaptically where they modulate neurotransmitter
release (Krivoy et al., 2008).

1.3 The Hypoglutamatergic Hypothesis of Schizophrenia

The first report of glutamate involvement in schizophrenia came almost 40 years ago,
with reduced levels of glutamate in the cerebrospinal fluid of patients with
schizophrenia (Kim et al., 1980). Whilst these initial reports were unable to be replicated
(Korpi et al., 1987; Perry, 1982), the hypothesis that glutamate is implicated in the
pathology of schizophrenia, is still at the forefront of schizophrenia research, due to an
array of evidence from genetic, imaging, postmortem and animal studies.

Hypofunction of the glutamatergic system is widely believed to be a driving force

underlying the pathology of schizophrenia, at least in a subset of patients. In particular,
the concept of NMDA receptor deficits has remained at the forefront of the hypothesis.
As previously mentioned (Section 1.1.1), no single gene has been found to be
responsible for schizophrenia, however several genes encoding the NR1 (GRIN1), NR2A
(GRIN2A) and NR2B (GRIN2B) subunits of the NMDA receptor have been associated with
the disorder (Demontis et al., 2011; Galehdari et al., 2009; Martucci et al., 2006;
Miyatake et al., 2002; Qin et al., 2005; Tang et al., 2006). Furthermore, a variety of
candidate or high-risk genes have been associated with schizophrenia, such as NRG1
(Bakker et al., 2004; Fukui et al., 2006; Munafò et al., 2006; Stefansson et al., 2002;
Williams et al., 2003), DISC1 (Callicott et al., 2005; Millar et al., 2000, 2001) and DTNBP1
(Schwab et al., 2003; Straub et al., 2002; Williams et al., 2004). Neuregulin 1, DISC1 and
dysbindin have been demonstrated to directly or in-directly regulate the glutamatergic

Jeremy Lum 26
system, in particular NMDA receptor activity. Neuregulin 1 and DISC1 co-localise with
NMDA receptors in the post-synaptic density and their respective activity has been
shown to regulate the expression and activity of the NMDA receptor (Bjarnadottir et al.,
2007; Hahn et al., 2006; Wei et al., 2014). In addition, dysbindin interacts with proteins
responsible for synaptic glutamate release and may consequently influence NMDA
receptor function (Saggu et al., 2013). Collectively, these studies suggest dysfunction of
the NMDA receptor may be a convergence point for the various genes associated with
schizophrenia (Snyder and Gao, 2013; Banjeree et al, 2010).

Complimentary to genetic studies, investigations of the NMDA receptor in human

postmortem studies support NMDA receptor abnormalities in schizophrenia. The
majority of studies have focused on the prefrontal cortex and hippocampus, with several
studies reporting reduced NR1 mRNA and/or protein expression in these two regions
(Beneyto and Meador-Woodruff, 2008a; Errico et al., 2013; Hahn et al., 2006; Weickert
et al., 2013), although it should be noted there are also reports of no changes in other
cohort studies (Emamian et al., 2004; Henson et al., 2008; Kristiansen et al., 2006;
Kristiansen et al., 2010; Le Corre et al., 2000) and an isolated report of increased NR1
mRNA expression in an elderly cohort (Dracheva et al., 2001). Findings regarding the
NR2 subunits in schizophrenia are inconsistent, with reports of increased, decreased and
no change in NR2A and NR2B mRNA or protein expression in the prefrontal cortex and
hippocampus (for detailed review see Geddes et al., 2011). In subcortical regions such
as the nucleus accumbens, studies investigating NMDA receptor subunit mRNA
expression and binding have reported no differences between schizophrenia and
unaffected subjects (Aparicio-Legarza et al., 1998; Meador-Woodruff et al., 2001; Noga
et al., 1997). However, no study to date has examined the protein expression of NMDA
receptor subunits in the nucleus accumbens, despite recent reports suggesting
glutamatergic alterations in this region (discussed in Section 1.1.2)(McCollum et al.,
2015; McCollum and Roberts, 2015). Collectively, postmortem results can be conflicting
dependent on the study, brain-region and target variable (mRNA or protein).
Discrepancies within the same brain region could reflect cohort specific factors, in
addition to technical factors across laboratories. Furthermore, it is widely accepted that

Jeremy Lum 27
mRNA levels do not generally indicate protein levels, which may explain differences
between mRNA and protein measures (Greenbaum et al., 2003). Whilst a clear trend
does not seem apparent, a recent meta-analysis of NR1 mRNA and protein expression
in the cortex of schizophrenia postmortem studies indicated a significant reduction in
schizophrenia (Catts et al., 2016). Furthermore, the authors concluded a sample size of
30 subjects per group is required to detect statistical significance based on the effect
sizes, at least in the cortex, suggesting many previous studies, which used sample sizes
as low as 6 per group may be underpowered (Catts et al., 2016).

One of the strongest pieces of evidence supporting the NMDA receptor hypofunction
hypothesis stems from the observation that NMDA receptor antagonists such as
phencyclidine (PCP) and ketamine induce positive, negative and cognitive
schizophrenia-like symptoms in healthy human subjects (Cohen et al., 1962; Cosgrove
and Newell, 1991; Javitt and Zukin, 1991; Lahti et al., 1995). Furthermore, these
compounds can exacerbate these symptoms in schizophrenia subjects (Javitt and Zukin,
1991). Even at relatively low doses, PCP can produce psychotic and progressive
withdrawal similar to the negative symptoms associated with schizophrenia (Luby et al.,
1959). Chronic recreational users of PCP are reported to show impaired cognitive
performance, similar to those associated with schizophrenia (Cosgrove and Newell,
1991; Javitt and Zukin, 1991). Furthermore, PET imaging studies of chronic recreational
PCP users suggests neuroanatomical similarities with schizophrenia subjects
(Hertzmann et al., 1990). Collectively, these clinical observations suggest NMDA
receptor blockade produces behaviours similar to schizophrenia and may provide insight
into its underlying pathology.

Ensuing from clinical observations of the effects of NMDA receptor antagonist

administration, in particular PCP, a field of research was dedicated to establish an animal
model of relevance to schizophrenia. In adult rodents, acute PCP treatment causes
behaviours analogous with schizophrenia such as hyperlocomotion, social withdrawal,
cognitive impairment and deficits in pre-pulse inhibition (Egerton et al., 2005; Kalinichev
et al., 2008; Mansbach and Geyer, 1989; Sams-Dodd, 1995). Furthermore, acute

Jeremy Lum 28
injection of PCP has been shown to increase dopamine release within the striatum
(nucleus accumbens), a characteristic associated with schizophrenia pathology (Carboni
et al., 1989). However, acute PCP treatment does not recapitulate the chronic manner
or neurodevelopmental trajectory associated with schizophrenia.

Repeated administration of PCP to rodents, generally including twice-daily PCP

administration for 7-14 days, with a 7-day washout period before testing, in adult
rodents has shown to produce behaviours analogous to schizophrenia. A major
advantage of the PCP model is its ability to recapitulate negative symptoms, which are
not observed in amphetamine models. Chronic PCP treatment has been shown to induce
social behaviour deficits (Lee et al., 2005; Qiao et al., 2001; Sams-Dodd, 1996, 1998).
Furthermore, social withdrawal deficits have been shown to be reversed by treatment
with the atypical antipsychotic, clozapine (Qiao et al., 2001; Sams-Dodd, 1998). In
addition, many cognitive behavioural impairments have been observed following
chronic PCP treatment, including deficits in reversal learning (Abdul-Monim et al., 2006;
Idris et al., 2010; McLean et al., 2010), novel object recognition tasks (Grayson et al.,
2007; Idris et al., 2010), episodic memory (Le Cozannet et al., 2010), attentional set
shifting (Broberg et al., 2009; Goetghebeur and Dias, 2009), spatial learning and memory
(Beraki et al., 2008; Jentsch et al., 1997). In fact, chronic PCP administration may better
mimic the schizophrenia pathology compared to acute treatment, as behaviours are
observed days or weeks following cessation of PCP treatment.

Acute and chronic administration of PCP in adult rats induces schizophrenia-like

behaviour. However, a limitation to these models is they do not possess the construct
validity for the neurodevelopmental hypothesis of schizophrenia. The concept of the
neurodevelopmental hypothesis suggests that early insult, may give rise to the
pathology of schizophrenia. Considering, the critical role of the NMDA receptor in
neurodevelopment, and the fact environmental insults and candidate genes associated
with schizophrenia (NRG1, DISC1 and DTNBP1) converge to disrupt NMDA receptor
activity during development it is conceivable that early insult to NMDA receptor
expression or activity during critical stages of neurodevelopment may contribute to the

Jeremy Lum 29
pathogenesis of schizophrenia. In an effort to address this, a perinatal PCP rat model
was established, whereby pups are treated with PCP (10 mg/kg) on PN7, 9 and 11 (Wang
et al., 2001). This model is the most widely used perinatal PCP model and has been
extensively characterised with long lasting behavioural and molecular deficits that are
associated with schizophrenia.

PCP administration (10 mg/kg) at PN7, 9 and 11 has repeatedly demonstrated to cause
pre-pulse inhibition deficits at adolescence (PN24-28) (Anastasio and Johnson, 2008a;
Wang et al., 2001, 2003). Furthermore, perinatal PCP treatment induces locomotor
sensitisation at adulthood following an acute PCP injection (Anastasio and Johnson,
2008a; Wang et al., 2001). In addition, pre-pulse inhibition deficits and PCP-induced
hyperlocomotive behaviour have been attenuated with acute antipsychotic treatment,
demonstarting predictive validity in this model (Anastasio and Johnson, 2008a).
Moreover, cognitive deficits have been repeatedly reported in adult rats treated with
PCP during the perinatal period, including deficits in social recognition, cognitive
flexibility in set-shifting tasks and acquisition and performance of spatial learning tasks
(Broberg et al., 2009; Depoortère et al., 2005; Harich et al., 2007; Wiley et al., 2003).
Furthermore, reduced performance in Morris water maze tasks has been observed in
this model, which was attenuated with chronic treatment of the glutamate co-agonist,
D-serine, suggesting that reduced glutamatergic signalling may underpin the cognitive
deficits in this model (Andersen and Pouzet, 2004). In line with this, altered NR1 and
NR2A subunit expression has been reported in the frontal cortex and striatum (Anastasio
and Johnson, 2008b, 2008a; du Bois et al., 2012; Wang et al., 2001). Reports from our
laboratory have shown that this model also produces alterations in NMDA and GABAA
receptor binding in the frontal cortex, hippocampus and thalamus, throughout critical
stages of neurodevelopment, suggesting an imbalance of the major excitatory and
inhibitory neurotransmitter systems (du Bois et al., 2009). Furthermore, deficits in
neuregulin1/erbB4 signalling, a major regulator of excitatory/inhibitory
neurotransmitter tone and a pathway implicated in the pathology of schizophrenia have
been observed in various brain regions of rodents treated with PCP during the perinatal
period (du Bois et al., 2012). This model has also shown changes to the dopaminergic

Jeremy Lum 30
system, with reports indicating developmental changes to D2 receptor binding and
expression of the dopamine synthesis enzyme, tyrosine hydroxylase (du Bois et al.,
2008). Collectively, the aforementioned studies suggest perinatal PCP treatment
produces behavioural and neurochemical changes associated with schizophrenia
pathology. Furthermore, these abnormalities are present weeks and months following
the initial insult period, indicating permanent developmental changes to the structure
and circuitry of the brain. Therefore, perinatal antagonism of the NMDA receptor
produces a valuable model in which to study the neurodevelopmental and
hypoglutamatergic hypothesis of schizophrenia.

Collectively, there are several converging lines of evidence implicating reduced NMDA
receptor activity in schizophrenia. Based on this evidence, exploring avenues to improve
NMDA receptor activity has become a highly attractive area of research. In line with this,
treatment with co-agonists such as glycine, D-serine and D-cycloserine have shown
promise in the management of schizophrenia symptoms (Labrie and Roder, 2010).
Furthermore, the potential of mGluRs to modulate NMDA receptor activity has been
identified as a possible treatment opportunity (Vinson and Conn, 2012); group 1 mGluRs
are of particular interest as they share a physical interaction with the NMDA receptor.
Group 1 mGluRs form physical associations with the NMDA receptor in various brain
regions including the hippocampus, prefrontal cortex and striatum, regions strongly
associated with schizophrenia pathology (Alagarsamy et al., 2002; Henry et al., 2002;
Luccini et al., 2007). Group 1 mGluRs and the NMDA receptor are co-localised on
postsynaptic membranes and their physical link is supported by the scaffolding proteins
Homer, SH3 and multiple ankyrin repeat domains (SHANK), guanylate-kinase-associated
protein (GKAP) and post-synaptic density 95 (PSD-95) (Tu et al., 1999). Furthermore, this
physical link provides a functional relationship, whereby mGluR1/5 activity indirectly
regulates NMDA receptor currents (discussed in Section 1.4.2). In addition, NMDA
receptor activity has been shown to modulate the activity of group 1 mGluRs
(Alagarsamy et al., 2005). However, this begs the question, if NMDA receptor activity is
implicated in the pathology of schizophrenia, do group 1 mGluRs also play a role and
provide a potential new avenue for treatment?

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1.4 Group 1 mGluRs

1.4.1 Expression and Splice Variants

Group 1 mGluRs are widely expressed throughout the CNS, however are most abundant
within the cortex, hippocampus, striatum and nucleus accumbens, brain regions
implicated in the pathology and treatment of schizophrenia (Kerner et al., 1997; Carmelo
Romano et al., 1996; Shigemoto et al., 1993, 1992). Whilst both mGluR1 and mGluR5
are expressed in neurons, only mGluR5 is expressed in glial cells, including
oligodendrocytes, neural stem cells, microglia and astrocytes (Byrnes et al., 2009; Luyt
et al., 2003; Romano et al., 1996). Furthermore, mGluR1 and mGluR5 are both
predominately expressed post-synaptically, however there is evidence that mGluR1 and
mGluR5 may additionally be expressed pre-synaptically and modulate glutamate release
(Gereau Iv and Conn, 1995; Mannaioni et al., 2001).

mGluR1 and mGluR5 are encoded by the GRM1 and GRM5 genes, respectively, and can
undergo alternate splicing. Splicing of the mGluR1 mRNA, gives rise to 5 distinct C-
terminal variants: mGluR1a-e; however in rats an additional splice variant mGluR1f has
been identified which produces an identical protein to mGluR1b (Hermans and Challiss,
2001). mGluR1a and mGluR1b are the most abundant splice variants, with mGluR1b
displaying a truncated C-terminal and slower Ca2+ kinetics, than the mGluR1a variant
(Pin et al., 1992). Meanwhile, mGluR5 consists of two primary splice variants, mGluR5a
and mGluR5b, with mGluR5b containing a 32 amino acid insert within the C-terminus.
Pharmacological studies show negligible differences between mGluR5a and mGluR5b
with both displaying similar functional characteristics to mGluR1a (Joly et al., 1995).

1.4.2 Signalling
mGluR1 and mGluR5 positively couple to Gq/11 and subsequent agonist-induced
stimulation causes activation of phospholipase C, consequently initiating
phosphatidylinositol 4,5-biphosphate (PIP2) hydrolysis into inositol 1,4,5 triphosphate
(IP3) and diacyl-glycerol (DAG). This process leads to IP3-mediated Ca2+ release from
intracellular stores and DAG-mediated activation of protein kinase C (PKC). Activation of
PKC can lead to a wide range of signalling processes, such as the activation of

Jeremy Lum 32
mammalian target of rapamycin (mTOR) pathways, cAMP response element-binding
protein (CREB) and brain-derived neurotropic factor (BDNF) production, signalling
molecules which have all been implicated in neuropsychiatric disorders (Niswender and
Conn, 2010). Furthermore, activation of PKC can stimulate proline-rich tyrosine
kinase/cell adhesion kinase (Pyk2/CAKβ) to recruit Src, which in turn can phosphorylate
tyrosine residues on the NMDA receptor, increasing channel open probability
(Groveman et al., 2012; Lu et al., 1999) (Figure 1.1). Whilst it was previously thought
mGluR5, not mGluR1, was responsible for potentiating NMDA receptor currents (Awad
et al., 2000; Doherty et al., 1997, 2000; Jia et al., 1998; Mannaioni et al., 2001; Pisani et
al., 2001), this is probably due to the fact the earlier investigated systems did not express
high levels of mGluR1, expressed alternative splice variants (mGluR1b/c, not mGluR1a)
or lacked co-localisation of mGluR1 and NMDA receptors. However, in CA3 (Cornu
Ammonis) neurons, where mGluR1 is highly expressed and co-localised with NMDA
receptors, mGluR1 has been shown to potentiate NMDA receptor currents (Benquet et
al., 2002). Furthermore, mGluR1 can also activate NMDA receptors via a G-protein
independent pathway, likely due to the binding of the adaptor protein, β-arrestin and
subsequent Src recruitment, which has previously been reported for other mGluRs
(Lefkowitz and Shenoy, 2005). In addition, there is evidence group 1 mGluRs may also
possess a capacity to activate alternate G-protein signalling pathways. mGluR1a was
shown to activate phosphoinositide hydrolysis and cAMP accumulation in transfected
Xenopus oocytes, Chinese hamster ovary (CHO), baby hamster kidney (BHK) and Human
embryonic kidney 293 (HEK293) cells (Aramori and Nakanishi, 1992; Francesconi and
Duvoisin, 1998; Hermans et al., 2000; Parmentier et al., 1998; Thomsen, 1996). Similarly,
mGluR5 was shown to stimulate cAMP accumulation in oocytes and Lilly Laboratories
Cell-Porcine Kidney (LLC-PK1) cells (Joly et al., 1995), but not in CHO cells, suggesting
possible coupling to the Gs protein-coupled receptor pathway (Abe et al., 1992).
Collectively, the evidence suggests mGluR1 and mGluR5 signalling is more complex than
previously thought and appears to depend on cell types, in addition to receptor
expression levels.

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Figure 1.1: Illustration of G-protein-dependent and independent potentiation of the
NMDA receptor. mGluR1 and mGluR5 are physically linked to the NMDA receptor
(NMDAR) via the scaffolding proteins Homer, SH3 and multiple ankyrin repeat domains
(SHANK), guanylate-kinase-associated protein (GKAP) and post-synaptic density 95
(PSD-95). Glutamate stimulated activation of mGluR1 or mGluR5 activates the positively
coupled G-protein, Gq/11, consequently stimulating phospholipase C (PLC) and
production of 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), causing release of
endoplasmic reticulum calcium (Ca2+) stores. A rise in intracellular calcium activates
protein-kinase C (PKC) and subsequently phosphorylates proline-rich tyrosine
kinase/cell adhesion kinase β (Pyk2/CAKβ) and consequently Src, which in turn
phosphorylates tyrosine residues of the NMDA receptor, increasing open channel
probability. Alternatively, mGluR1 can potentiation the NMDA receptor independent of
G-protein pathways, presumably via coupling to β-arrestin and subsequent Src
recruitment.

Whilst G-protein coupled receptors (GPCRs) are typically considered responsible for
transmitting extracellular stimuli to intracellular responses, many GPCRs are also found
on intracellular membranes. In addition to group 1 mGluR expression on the cell
membrane, they are also localised on the endoplasmic reticulum and nuclear membrane
(Hubert et al., 2001a; López-Bendito et al., 2002; Petralia et al., 1997). In fact, a large
proportion of mGluR1 (40-60%) and mGluR5 (60-90%) are located on intracellular
membranes, rather than the plasma membrane, depending on the brain region
investigated (Hubert et al., 2001; Lopez-Bendito et al., 2002; O’Malley et al., 2003).
O’Malley and colleagues have identified unique roles for group 1 mGluRs, dependent on
their cellular localisation, in particular the nuclear membrane, adding further complexity
to their signalling and function. Intracellular group 1 mGluRs are able to be activated by

Jeremy Lum 34
intracellular glutamate entering via ion dependent transporters/exchangers or released
via the endoplasmic reticulum (Jong et al., 2005; Kumar et al., 2008). Furthermore,
subsequent activation of nuclear mGluR1 and mGluR5 is capable of activating unique
Ca2+ signatures and signalling pathways to their cell surface counterparts. Specific
activation of intracellular mGluR1 or mGluR5 in cultured striatal neurons was shown to
result in phosphorylation of ERK1/2, Elk-1 and CAMKII with intracellular mGluR5
activation also causing upregulation of transcriptional factors involved in neuronal
growth, survival and synaptic plasticity (Jong et al., 2007; Jong et al., 2009). In addition,
using hippocampal slices, activation of intracellular mGluR5 was shown to play a role in
long-term depression (LTD), however not potentiation (Purgert et al., 2014).
Collectively, these studies highlight the unique signalling and functional consequences
of group 1 mGluRs, dependent on their subcellular localisation.

1.4.3 Neurodevelopmental Profile

Group 1 mGluRs are required for normal neurodevelopment with mGluR1 and mGluR5
knock-out (KO) mice showing various signalling and behavioural deficits (discussed later
in Section 1.6.1). mGluR1 and mGluR5 are primarily responsible for the high level of PIP2
hydrolysis and PKC activation during CNS development, which is required for cell growth
and differentiation (Berridge, 1993). Furthermore, group 1 mGluRs play a role in LTD
and long-term potentiation (LTP), the underlying processes involved in synapse
formation. Group 1 mGluRs, in particular mGluR5, have shown to be highly expressed
by neuroprogenitor cells where they play a role in the survival and proliferation of neural
stem cells within the subventricular zone and dentate gyrus (Di Giorgi Gerevini et al.,
2004; Di Giorgi-Gerevini et al., 2005). In line with this, mGluR1 and mGluR5 mRNA and
protein expression has been detected in the CNS of human and rodent embryos.
Postnatal mGluR1a expression in the CNS appears to increase from birth for the first two
weeks of rodent neurodevelopment, however reaches a plateau thereafter (Casabona
et al., 1997; Catania et al., 1994; Di Giorgi Gerevini et al., 2004)(Table 1.1). Whilst
mGluR5 expression appears to be abundant in the first week of postnatal development
in the rodent, it appears to plateau or decline at later adolescent and adult time points
(Casabona et al., 1997; Catania et al., 1994; Di Giorgi Gerevini et al., 2004; Romano et

Jeremy Lum 35
al., 1996). However, expression analysis showed this is likely due to a decline in the
mGluR5a splice variant following birth, with mGluR5b remaining stable throughout
postnatal stages (Romano et al., 2002). This may suggest there are functional differences
between the mGluR5a and mGluR5b splice variants in regards to their role in
neurodevelopmental processes. In line with this, homologous cells transfected with
these splice variants suggest mGluR5a hinders neuronal maturation, whilst mGluR5b
increases neurite growth (Mion et al., 2001), however little has been done to further
identify functional differences. Furthermore, it appears mGluR5 undergoes functional
signalling changes throughout the ageing process. Striatal slices collected from young
rats (3 months) exhibit higher mGluR5 protein levels than old (24 months) rats
(Domenici et al., 2003). In addition, this decline of mGluR5 protein expression is
associated with reduced functionality as aged rats displayed lower phosphoinositide
turnover in response to the mGluR5 specific agonist, CHPG, as well as decreased ability
to potentiate NMDA receptor mediated currents (Domenici et al., 2003). This age-
dependent decline of mGluR5 expression and signalling was also associated with a
decline in phospholipase C-β1, indicating an age dependent loss of mGluR5 signalling
partners. In line with this, Kaja and colleagues reported a change in the ratio of short
and long forms of Homer1, a multivalent complex responsible for the association of
mGluR5 and IP3 signalling, in the aged forebrain (Kaja et al., 2013). Collectively, these
results suggest group 1 mGluR expression and downstream signalling display changes
throughout postnatal development and ageing.

1.4.4 Structure
Group 1 mGluRs share similar primary, secondary, tertiary and quaternary structure.
Group 1 mGluRs possess a large N-terminus that encases a venus-fly-trap domain for
agonist binding (Parmentier et al., 1998). The N-terminus links to a 7-transmembrane
domain, which plays a role in activation of coupled G-proteins. Furthermore, group 1
mGluRs possess a C-terminus tail; this region exhibits the largest variation between
mGluR1 and mGluR5.

Jeremy Lum 36
Table 1.1: Summary of studies investigating the neurodevelopmental mRNA and protein expression profile of mGluR1 and mGluR5.
Species/ Analysis mRNA/
Age Reported findings Reference
Brain region Protein
Human Studies
Marginal zone, Gestational ages (GA): 9, Immuno-  mGluR1a expression was detected in cortical plate at all time-points (Boer et
cortical plate and 10, 13, 16, 17, 20, 22, 23, histochemistry:  mGluR1a expression was detected in the marginal zone at 36-40 weeks gestational age al., 2010)
ventricular zone 25, 29, 31, 36 and 40 weeks mGluR1a and and <7 months postnatal.
(n=1/time point) mGluR5  mGluR5 was strongly expressed in marginal zone, cortical plate and ventricular zone at
Postnatal ages: 3 weeks, 2 all gestational and postnatal time points
months, 7 months and 8
years (n=1/time point)

Temporal cortex Gestational ages: 13 weeks Western Blot:  mGluR1a and mGluR5 expression were highest at 13 weeks gestation and decreased at
Postnatal ages: 2 months mGluR1a and 2 months postnatal, where it was similar at 30 years.
and 30 years (n=1/time mGluR5 (monomer)
point)
Hippocampus Gestational ages: RT-PCR: pan-  mGluR5 mRNA significantly increased progressively with all ages (Yang et
9-11 weeks (n=9) mGluR5 al., 2012)
14-16 weeks (n=10)
2-24 weeks (n=9) Western blot:  mGluR5 protein significantly increased progressively with all ages
32-36 weeks (n=6) mGluR5 (monomer)
Rodent Studies
Sprague-Dawley rats; Postnatal day (PND) 1, 3, 7, In situ  Low level of mGluR1 expression at PND0 in all brain regions; expression gradually (Catania et
Cortex, striatum, 10, 14, 21, 30 and PND60 Hybridisation: pan- increased until PND21, where adulthood (PND60) levels were reached. al., 1994)
hippocampus (n=3-4/group) mGluR1 and pan-  mGluR5 was highly expressed in the cortex and striatum at PND1 and slowly declined
(dentate gyrus and mGluR5 over the first 3 weeks of postnatal development and remained constant until PND60.
internal granular  mGluR5 expression remained constant within the hippocampus at all time-points
layer) investigated
Wistar Rats; Cortex, PND3, 7, 14, 21, 28 and 35 RT-PCR: mGluR5a  mGluR5a was the abundant form in olfactory bulb at all time-points examined, (Minakami
hippocampus, and mGluR5b compared to mGluR5b et al.,
olfactory bulb  mGluR5a was predominant form at PND3 and 7 in cortex and hippocampus 1995)
 mGluR5b was predominant form from PND14-35 in cortex and hippocampus

Jeremy Lum 37
 Sprague-Dawley rats; PND7 and adult RT-PCR: mGluR5a  mGluR5a was higher at PND7 than adulthood. (Romano
Cortex, hippocampus, and mGluR5b  mGluR5b mRNA levels were similar at PND7 and adulthood. et al.,
hypothalamus, 1996)
striatum, olfactory PND0, 4, 7, 11, 18, 27 and Western blot:  mGluR5 protein expression (unable to differentiate between splice variants) increased
bulb and cerebellum 70 (adult) mGluR5 (monomer) in cortex, hippocampus, striatum, hypothalamus and brainstem from PND0-11 and
then decreased at PND18, where levels remained relatively stable until PND70.
 mGluR5 levels in the cerebellum at PND0 and decreased throughout the time-points
investigated
 mGluR5 levels in the olfactory bulb remained stable throughout time-points examined
Sprague-Dawley rats; PND9 and 2 months (adult) Western blot:  mGluR1a expression was higher at PND9 than adulthood in the hypothalamus, (Casabona
Cortex, hippocampus, mGluR1a and olfactory bulb and hippocampus. et al.,
striatum, olfactory mGluR5 (monomer)  mGluR1a expression was higher at in the adult cerebellum than at PND9 in the 1997)
bulb, cerebellum, hypothalamus, olfactory bulb and hippocampus.
hypothalamus  No difference in mGluR1a protein levels were found between PND9 and adult corpus
striatum and cerebral cortex
 mGluR5 protein expression was greater at PND9 than adulthood in all brain regions.
Sprague-Dawley rats; PND7 and adult Western blot:  mGluR5b protein levels were higher at adulthood than PND7 in the cortex and (Romano
Cortex, hippocampus, mGluR5b hippocampus. et al.,
hypothalamus, (monomeric band)  mGluR5b protein levels were similar at PND7 and adulthood in the hypothalamus and 2002)
striatum, olfactory striatum.
bulb and cerebellum  mGluR5b levels lower at adulthood than PND7 in the brainstem, cerebellum and
olfactory bulb
Sprague-Dawley rats; Gestational day (GD) 12, 15 RT-PCR: mGluR1a/b  mGluR1a/b mRNA was detected at GD12. (Di Giorgi
Cortex, hippocampus and PND1, 10, 21 and mGluR5a  mGluR1a/b mRNA increased from PND1-21 in the cortex and cerebellum, whilst in the Gerevini et
and cerebellum hippocampus expression increased from PND1-10 and decreased between PND10-21. al., 2004)
 mGluR5a: Detected in cortex and hippocampus at all time-points investigated.
Western blot:  mGluR1a protein expression was undetectable during embryonic development.
mGluR1a (dimer  mGluR1a protein was detected at PND1 in all brain regions and increased with age in
and monomer) and the cortex and cerebellum. However, in the hippocampus mGluR1a protein expression
mGluR5 (monomer increased between PND1-10 and then decreased between PND10-21.
only)  mGluR5 protein expression was detectable during embryonic development and
expression peaked between PND1-10, but then declined at PND21, in all brain regions
examined.
Abbreviation: RT-PCR, real-time polymerase chain reaction; GA, gestational age, GD, gestational day; PND, postnatal day.

Jeremy Lum 38
In addition to possessing the venus-fly trap domain, which acts as a site for agonist
binding, the N-terminus of group 1 mGluRs contains cysteine residues capable of
forming disulphide bridges and consequent homo- and hetero-dimers, a characteristic
which appears to be conserved across all mGluRs (Romano et al., 2001). Recent studies
suggest mGluR1 and mGluR5 are capable of heterodimerisation with each other,
however not the group 2 or 3 family of mGluRs (Doumazane et al., 2011). In addition,
mGluR5 may also be capable of dimerisation with the adenosine A2A and dopamine, D2
receptors (Fuxe et al., 2014; Popoli et al., 2001). The existence of mGluR1 and mGluR5
homodimers was reported almost 20 years ago. Romano and colleagues first observed
an mGluR5 band that was twice as large as its predicted molecular weight, under non-
reducing western blot conditions (Romano et al., 1996). The addition of reducing agents,
dithiolthreitiol or 2-mercaptoethanol caused mGluR5 to migrate at its apparent
molecular weight, indicating the breakdown of mGluR5 covalent disulphide bridges. In
addition, treatment of the tissue with iodeoacetamide, which alkylates free sulfhydryls
(preventing the ex vivo formation disulphide bonds), did not change appearance of
these higher molecular weight species, indicating these species were not spontaneously
forming postmortem. It is now clear mGluR1 and mGluR5 form homodimers within the
endoplasmic reticulum and Golgi apparatus before transportation to various cellular
locations such as the plasma, mitochondrial or nuclear membrane (Pin et al., 2003;
Sergin et al., 2017). Collectively, these findings clearly show mGluR1 and mGluR5 are
capable of homodimerisation and are natively expressed in this manner. Furthermore,
dimerisation appears to be vital for agonist-induced signalling. El Moustaine and
colleagues (2012) demonstrated although dimerisation of mGluRs is not required for G-
protein coupling, dimerisation is necessary for agonist-induced activation, rendering the
monomeric form inactive to agonist-induced activation (Figure 1.2). In addition, it was
demonstrated that binding of a positive allosteric modulator to a monomeric mGluR
caused the subsequent activation of the G-protein in the absence of glutamate (El
Moustaine et al., 2012). Furthermore, Kniazeff et al., (2004) demonstrated that whilst
binding of glutamate to an mGluR5 protomer (monomeric subunit of dimeric complex)
was sufficient to partially activate the receptor, ligand binding to both protomers
consequently caused full activation. In light of this evidence, examining these receptors

Jeremy Lum 39
in their functional dimeric state may provide a more complete picture of group 1 mGluR
signalling.

Figure 1.2: Schematic of proposed activation of dimeric and monomeric mGluRs by

orthosteric agonists and positive allosteric modulators. a. Binding of glutamate to a
dimeric mGluR causes G-protein activation. b. Binding of a positive allosteric modulator
to a dimeric mGluR in the absence of glutamate does not cause G-protein activation and
c. requires the presence of glutamate, for subsequent G-protein activation. d.
Glutamate binding to a monomeric mGluR does not result in G-protein activation,
however e. a positive allosteric modulator can result in G-protein activation in the
absence of glutamate.

Group 1 mGluRs contain a 7-transmembrane domain, which has become of recent

interest for drug discovery efforts. A large effort has focused on identifying compounds
with allosteric modulatory properties for group 1 mGluRs, in particular mGluR5.
Allosteric modulators bind in an alternate location (allosteric site) to the traditional
orthosteric site (i.e. glutamate binding site), changing the conformation of the receptor
and consequently up or down regulating the activation of the receptor by its orthostertic
ligand. Allosteric modulators can alter the pharmacological characteristics of a receptor
in a variety of ways, including increasing or decreasing the affinity of the orthosteric
ligand or downstream signalling pathways of orthosteric ligands, or alter receptor
activity, irrespective of orthosteric ligand binding (Christopoulos and Kenakin, 2002).
There are several allosteric sites found within the 7-transmembrane region of group 1
mGluRs, (Chen et al., 2008; O’Brien et al., 2003; Wu et al., 2014), providing many drug
targets. Allosteric sites provide several key advantages over traditional orthosteric

Jeremy Lum 40
ligands. One major advantage is that orthosteric sites tend to be conserved across
receptor families; therefore, allosteric ligands provide greater target receptor specificity
than their traditional orthosteric counterparts. Furthermore, allosteric modulators
which do not display agonist activity rely on endogenous ligand activity, avoiding
desensitisation or toxic effects observed with orthosteric ligands (Conn et al., 2009).

The C-terminus of mGluR1 and mGluR5 contains multiple sites in which a multitude of
proteins can directly interact and regulate receptor activity, signalling and localisation.
As previously mentioned, mGluR1a and mGluR5a/b exhibit the longest C-terminus of
the group 1 mGluR splice variants and are the most abundantly expressed in the brain.
The longer C-terminus of these splice variants provides a region for direct protein
interactions with various protein kinases and phosphatases. PKC is the most extensively
investigated kinase associated with group 1 mGluRs and has been demonstrated to
constitutively phosphorylate group 1 mGluRs. In addition, PKC is responsible for
phosphorylation of mGluR1 and mGluR5 at several serine and threonine sites along the
C-terminus upon agonist stimulation. Similar to many other G-protein coupled
receptors, PKC-dependent phosphorylation of group 1 mGluRs exhibits a negative
feedback loop to promote desensitisation, in response to prolonged agonist-induced
stimulation. In addition, G-protein receptor kinase 2 (GRK2) has also shown to play a
role in mGluR1/5 desensitisation via coupling the receptor to β-arrestin and
consequently mediating clathrin coated endocytosis pathways. Furthermore, PKC
phosphorylation of sites such as T695 and T681 are thought to reduce G-protein
coupling and consequently reduce downstream group 1 mGluR signalling. It also appears
that group 1 mGluR signalling is tightly regulated via Ca2+ sensitive molecules, as
calmodulin (CaM) is capable of binding to PKC phosphorylation sites and prevent PKC
phosphorylation. Although not as extensively investigated as serine and threonine
phosphorylation, it has also been demonstrated that mGluR5 can undergo
phosphorylation at its tyrosine residues in cultured striatal neurons. NMDA receptor
activation has been shown to increase mGluR5 tyrosine residue phosphorylation,
highlighting the important role the C-terminus of group 1 mGluRs has in receptor cross
talk.

Jeremy Lum 41
Collectively, these interactor proteins of the group 1 mGluR C-terminus can regulate
cellular localisation, protein-protein association and internalisation. The family of
Homer1 and Norbin proteins also interact with group 1 mGluRs. Furthermore, they have
recently been implicated in the pathophysiology of schizophrenia and are of relevance
to the present thesis. The association of Homer1 and Norbin with group 1 mGluRs and
their relevance to schizophrenia and other neuropsychiatric disorders is reviewed
below.

1.5 Group 1 mGluR Interacting Partners-Homer1 and Norbin

1.5.1 Homer1
Brakeman and colleagues were the first to identify a group 1 mGluR endogenous
regulator, in the form of Homer1, which contains a PDZ-domain for interaction with the
C-terminus of group 1 mGluRs (Brakeman et al., 1997). Homer1 has three isoforms,
dependent on gene splicing; Homer1a (vesl-1S) is the shortest of the splice variants (186
amino acids) and is encoded by an immediate early gene. In addition to the short
Homer1a isoform, Homer1 can also be alternatively spliced into the Homer1 long forms
(vesl-1L); Homer1b and Homer1c (Shiraishi-Yamaguchi and Furuichi, 2007).

Homer1 expression is mainly localised to glutamatergic excitatory synapses and

regulated in response to synaptic activity. Homer1a expression is induced following
synaptic activity in the form of LTP, synaptogenesis, ischemia and seizures (Brakeman et
al., 1997; Kato et al., 1998). Meanwhile the long Homer1 isoforms are constitutively
expressed. Homer1 short and long forms share a common conserved enabled/VASP
homology 1 (EVH1) domain structure, which is capable of direct interaction with group
1 mGluRs (Brakeman et al., 1997). Furthermore, the long forms of Homer1 possess a
carboxyl-terminal domain or multimerisation domain and contain a coiled-coil structure
capable of forming homomeric or hetromeric interactions with a range of binding
partners (Tadokoro et al., 1999; Xiao et al., 1998). One major binding partner of the long
forms of Homer1 is the IP3 receptor, located on the membrane of the endoplasmic
reticulum. Homer1b/c mediates the association of group 1 mGluRs and the IP3 receptor

Jeremy Lum 42
to increase Ca2+ release from intracellular stores of (Tu et al., 1998). As Homer1a lacks a
coiled-coil domain, it prevents facilitation of this process. Furthermore, Homer1a
competitively binds to group 1 mGluRs to reduce this process, thereby acting in a
dominant negative manner (Xiao et al., 1998). It is clear that Homer1 plays an important
role in group 1 mGluR signalling and Ca2+ homeostasis and should be considered in this
context when assessing mGluR function.

1.5.2 Norbin/Neurochrondrin
Norbin (rodent) or neurochondrin-1 (human) is one of the most recently identified
endogenous regulators of group 1 mGluRs. Norbin is expressed in chondrocytes,
osteoblasts and osteocytes, however in the CNS, it is thought to localise specifically to
neurons (Ishiduka et al., 1999). Norbin is a cytosolic protein, widely distributed in
excitatory dendritic spines at the post-synaptic density and co-localised with group 1
mGluRs (Wang et al., 2009). A large majority of studies investigating Norbin have
focused on its association with mGluR5, with no further investigation of its role with
mGluR1, despite confirmation of a physical interaction. Norbin has been demonstrated
to play a role in mGluR5 cell surface expression, with neuronal cultures overexpressing
Norbin exhibiting increased surface mGluR5 (Wang et al., 2009). Furthermore, Norbin
potentiates mGluR5 signalling; HEK293 cells transfected with Norbin exhibit increased
mGluR5-stimulated inositol trisphosphate, ERK1/2 phosphorylation and intracellular
Ca2+ release (Wang et al., 2009).

Norbin is a relatively recently discovered protein and there are limited studies regarding
its function. Deletion of Norbin in rodents causes death during early embryonic periods,
indicating its importance in neuronal development (Mochizuki et al., 2003). Norbin
mRNA expression in the mouse embryo can be detected as early as embryonic day (ED)
10 where it is localised to the hindbrain (Mochizuki et al., 2003). mRNA and protein
expression within the cortex has been detected at ED18, progressively increasing until
postnatal day 21, which coincides with the period of neurite outgrowth (Mochizuki et
al., 2003). In line with this, overexpression of Norbin in Neuro2A cells increases neurite
number and length. Furthermore, Norbin expression is upregulated following

Jeremy Lum 43
chemically-induced LTP, the process underlying neural plasticity and memory formation
(Shinozaki et al., 1997). Additionally, conditional Norbin KO mice exhibit LTP deficits
(later discussed in Section 1.6.4). Despite the discovery of Norbin’s role in group 1 mGluR
signalling and neurite growth, there has been very little investigation of Norbin in
schizophrenia.

1.6 Group 1 mGluRs and their endogenous regulators in the pathology of

schizophrenia

1.6.1 Group 1 mGluRs-Evidence from knock-out and pharmacological models

Knockout models support the involvement of group 1 mGluRs in the pathophysiology of
schizophrenia. mGluR1 and mGluR5 KO mice exhibit sensorimotor deficits, a common
phenotype observed in schizophrenia patients (Brody et al., 2003, 2004). In addition,
hyperlocomotive deficits are also a common feature of schizophrenia rodent models,
particularly when challenged with dopaminergic or glutamatergic agents such as
amphetamine, phencyclidine, ketamine or MK-801. mGluR1 KO mice were first reported
to exhibit reduced locomotor activity, owing to severe motor deficits observed in these
mice, likely due to the role mGluR1 plays in cerebellar dysfunction (Conquet et al., 1994).
However, a later study reported that whilst fine motor activity was disturbed in mGluR1
KO mice, there was no change in baseline motor activity (Mao et al., 2001). Furthermore,
mGluR1 KO mice showed increased amphetamine-induced hyperlocomotor activity
compared to wild-type (WT) controls. mGluR5 KO mice exhibit basal locomotor activity
similar to WT controls (Chiamulera et al., 2001; Lu et al., 1997). Furthermore,
administration of the dopamine reuptake inhibitor, cocaine, which induces a
hyperlocomotive locomotor response in WT mice, has no effect in mGluR5 KOs, despite
causing similar increases of dopamine levels in nucleus accumbens (Chiamulera et al.,
2001). This study not only demonstrated that mGluR5 plays a role in cocaine-induced
behaviours, but highlights the important interaction between mGluR5, subcortical
dopaminergic system and associated behaviours. In addition, Gray and colleagues
(2009) reported mGluR5 KO mice exhibited increased hyperlocomotor activity in
response to the NMDA receptor antagonist, MK-801, compared to WT controls,
confirming the interaction that mGluR5 plays with the NMDA receptor. Collectively,

Jeremy Lum 44
these studies implicate a role for mGluR5 in the function of dopaminergic and
glutamatergic neurotransmission, key systems involved in the pathophysiology of
schizophrenia.

LTD and LTP are the molecular processes underlying synaptic plasticity and memory
formation, and are believed to be disrupted in schizophrenia, at least in a subset of
patients (Bhandari et al., 2016). An array of studies show group 1 mGluRs play an
integral role in synaptic plasticity and cognition, which has been clearly highlighted using
mGluR1 and mGluR5 KO models. mGluR1 KO mice exhibit memory acquisition and
retention deficits. Furthermore, these mice display reduced hippocampal LTP (Aiba et
al., 1994; Bordi et al., 1997). mGluR5 KO mice exhibit impaired hippocampal dependent
spatial learning performance in the Morris water maze and contextual fear conditioning
tasks (Jia et al., 1998). In line with this, mGluR5 KO mice display NMDA-dependent LTP
deficits in CA1 (Lu et al., 1997). Furthermore, intracerebroventricular injection of the
mGluR5 antagonist, MPEP, dose-dependently inhibited LTP in the CA1 and dentate gyrus
region of Wistar and Hooded Listar rats and impaired working and reference memory in
Wistar rats in radial maze tasks (Manahan-Vaughan and Braunewell, 2005). mGluR5 has
similarly shown to have a role in LTD, with LTD being abolished in mGluR5 KO mice
(Huber et al., 2001). Furthermore, Purgert and colleagues, employed a pharmacological
approach to activate cell surface and intracellular mGluR5, and identified that
intracellular mGluR5 is responsible for mediating electrically- and chemically-induced
LTD (Purgert et al., 2014). Induction of LTD via the group 1 mGluR agonist, (S)-3,5-
Dihydroxyphenylglycine (DHPG), was inhibited by the cell permeable mGluR5
antagonist, 2-Methyl-6-(phenylethynyl)pyridine (MPEP), which is able to bind to mGluR5
located not only on the cell surface but also on intracellular membranes such as the
endoplasmic reticulum and nuclear membranes. However, application the non-
permeable mGluR5 antagonist (capable of only inbiting cell surface mGluR5), LY53, was
unable to inhibit DHPG-induced LTD. This evidence indicates mGluR5’s role in LTD may
be regulated by specific pools of mGluR5. Collectively, these studies demonstrate a role
for group 1 mGluRs underlying synaptic plasticity and cognition; processed associated
with the pathology of schizophrenia.

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1.6.2 Group 1 mGluRs-Evidence from postmortem studies
The investigation of group 1 mGluR ligand binding, mRNA and protein levels in
schizophrenia subjects has previously been investigated across several postmortem
cohorts and brain regions. A majority of gene expression studies have reported
unaltered mGluR1 and mGluR5 transcript expression in the prefrontal cortex,
hippocampus, thalamus, striatum and cerebellum of schizophrenia subjects (Table 1.2).
However, there are also reports of altered expression, with Volk et al (2010) reporting a
12% increase in mGluR1a transcript expression in the prefrontal cortex (Brodmann Area
[BA] 9) of schizophrenia subjects and Fatemi and colleagues (2013) reporting reduced
mGluR5 mRNA expression in the lateral cerebellum of schizophrenia subjects.

Early studies examining mGluR1 and mGluR5 protein via immunoblot (under reducing
conditions) in schizophrenia postmortem cohorts have largely reported no change in
expression in the prefrontal cortex, caudate, putamen and nucleus accumbens (Table
1.2). It is worth considering that these studies were limited by small sample sizes (<20
samples). Despite this, a 10% increase of mGluR1a protein expression was reported in
the prefrontal cortex of schizophrenia subjects (Gupta et al., 2005). Furthermore, a
reduction of mGluR5 monomer protein has been observed in the lateral cerebellum and
prefrontal cortex (BA9) of schizophrenia subjects, however no change in the dimeric
form (Fatemi et al., 2013). Interestingly, our group previously investigated mGluR5
protein expression in a schizophrenia cohort that had shown evidence of NMDA
receptor dysfunction (Weickert et al., 2013). Whilst we found no change in mGluR5
protein levels (monomeric band only) within the dorsolateral prefrontal cortex of
schizophrenia subjects (Matosin et al., 2013), upon re-analysis of images and
quantification of the dimeric band, we found overall and dimeric mGluR5 protein levels
to be significantly increased within the same cohort. This highlights the importance of
examining dimeric levels in particular, as these are believed to be the canonical form of
mGluR5 receptors that reach the cell surface and interact with the NMDA receptor.
Furthermore, as mGluRs exist in dimeric and monomeric forms, without quantification
of both these bands, a true picture of mGluR protein expression cannot be reached.
Recently, we also reported dimeric and monomeric mGluR5 protein levels to be

Jeremy Lum 46
increased and mGluR1a levels to be decreased, in the CA1 region of the hippocampus,
when examined under reducing conditions (Matosin et al., 2016a, 2015c). Further
supporting the importance and implication of mGluR dimerisation, Corti et al (2007)
reported that whilst total (dimer and monomer) and monomeric levels of mGluR3 were
not altered in the prefrontal cortex (BA10), there was a significant reduction in mGluR3
dimeric levels. There is emerging evidence of dimeric alterations of group 1 mGluRs in
schizophrenia, however 1) no study has examined group 1 mGluR protein expression
under non-reduced conditions, so are not able to measure total dimeric levels; and 2) it
is unclear how widespread mGluR changes are in the brain.

In line with the glutamatergic hypothesis, the prefrontal cortex and hippocampus are
highly implicated in schizophrenia and are the focus of many postmortem investigations.
The prefrontal cortex and hippocampus are involved in short and long-term cognitive
function, both processes disrupted in schizophrenia pathology. These regions send
excitatory glutamatergic projections to the ventral striatum, in particular the nucleus
accumbens, another highly implicated region in schizophrenia (Britt et al., 2012). The
nucleus accumbens is involved in reward and motivation, comprising of medium spiny
neurons and dense with both dopaminergic and glutamatergic projections. It is thought
that current antipsychotics, primarily exert their therapeutic effect through D2 receptors
located on the medium spiny neurons (Deutch et al., 1992; Deutch and Cameron, 1992).
It is this convergence of glutamatergic and dopaminergic projections on a γ-
Aminobutyric acid (GABA) interface that makes the nucleus accumbens a region of vast
interest in schizophrenia pathology. Recently, evidence for glutamatergic pathology
within the nucleus accumbens was supported by a series of postmortem studies from
McCullum and Colleagues. McCollum and Roberts (2015) reported increased protein
expression of the vesicle glutamate transporter, VGLUT2, in the nucleus accumbens of
schizophrenia subjects. Further adding support to this glutamatergic dysfunction in the
nucleus accumbens, they reported an increased number of asymmetric synapses,
indicative of glutamatergic projections (McCollum et al., 2015). Whilst these studies are
recent and were performed in small cohorts (n=13-20), they encourage further evidence
characterising the glutamatergic system within the schizophrenia nucleus accumbens.

Jeremy Lum 47
Table 1.2: Summary of mGluR1 and mGluR5 investigations in postmortem human brain tissue from schizophrenia patients.
Cohort Brain region(s) Analysis Reported results Reference
7 SCZ Prefrontal cortex In situ hybridisation: encoding Increased mGluR5 mRNA (28%) in layer III of BA11 in SCZ, but no (Ohnuma et al.,
10 CT (PFC; (BA9, 10 and for mGluR5 change in BA9 or 10. 1998)
11))
5 SCZ Hippocampus In situ hybridisation: encoding No change in panmGluR5 mRNA transcripts. (Ohnuma et al.,
6 CT (Dentate gyrus, for panmGluR5 2000)
Cornu Ammonis
(CA1),
Parahippocampal
gyrus)
12 SCZ Thalamus In situ hybridisation: No change in both mGluR1 and mGluR5 mRNA. (Richardson-
8 CT encoding for mGluR1 and Burns et al.,
mGluR5 2000)
16 SCZ PFC ((BA9, 11, 32, Immunoblot: analysis for mGluR1a protein levels were increased in the PFC (10%) in SCZ. (Gupta et al.,
9 CT 46), mGluR1a and mGluR5 No difference of mGluR1a and mGluR5 in other regions 2005)
Caudate-Putamen, (monomeric band-reducing examined.
Nucleus Accumbens conditions not stated)
37 SCZ Dorsolateral PFC Receptor autoradiography: No significant difference between diagnosis and CT in [3H] MPEP (Matosin et al.,
37CT [3H] MPEP binding to mGluR5 binding or mGluR5 protein levels. 2013)

Immunoblot (reducing
conditions): mGluR5
monomeric band
15 SCZ Anterior cingulate Receptor autoradiography: No significant difference between diagnosis and CT. (Matosin et al.,
15 MD cortex [3H] MPEP binding to mGluR5 2013)
15 BP
15 CT
12 MDNP
12 MDP
12 CT

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20 SCZ PFC (BA9) qRT-PCR: panmGluR5 Reduction in mGluR5 transcript expression in BP (23%). (Fatemi et al.,
19 BP 2013)
29 CT Immunoblot (reducing mGluR5 monomer was reduced in SCZ (32-53%) and BP (39-63%)
conditions): mGluR5 compared to CT.
monomeric band

15 SCZ Lateral cerebellum qRT-PCR: panmGluR5 A reduction in mGluR5 mRNA in SCZ (51%) and MD (47%)
14 MD compared to CT
15 BP
14 CT Immunoblot (reducing mGluR5 protein was reduced in SCZ (60-75%), BP (54-79%) and
conditions): mGluR5 MD (49-64%) compared to CT.
monomeric band
37 SCZ Dorsolateral PFC qRT-PCR: panmGluR5 mGluR5 mRNA levels were unaltered. (Matosin et al.,
37 CT 2015a)
Immunoblot (reducing Total mGluR5 protein levels were increased (22%) in SCZ (dimer: -
conditions): mGluR5 dimeric 54%).
band
20 SCZ Hippocampus (CA1) Immunoblot (reducing Total mGluR5 protein levels were increased (42%) in SCZ. (Matosin et al.,
20 CT conditions): mGluR5 dimeric 2015c)
and monomeric bands Increase of mGluR5 dimer (52%) and monomer (25%) bands.
20 SCZ Hippocampus (CA1) Immunoblot (reducing Total mGluR1α protein levels were reduced (-32%) in SCZ. (Matosin et al.,
20 CT conditions): mGluR1a dimeric 2016a)
and monomeric bands Reduction in mGluR1a dimer (-66%) and monomer (-67%).
Abbreviations: SCZ, schizophrenia; CT, control; MD, major depressive disorder; BP, bipolar disorder; CA, Cornu Ammonis;, PFC, prefrontal cortex; BA,
Brodmann’s area.

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1.6.3 Homer1
Homer1 KO mice display schizophrenia-like behaviours, including increased locomotive
response when challenged with acute administration of MK-801 and methamphetamine
(Szumlinski et al., 2005). In addition, Homer1 KO mice display anxiety-like behaviour and
behavioural despair, as well as working and spatial memory deficits, which are mediated
by cortical and hippocampal regions, respectively (Gerstein et al., 2012; Szumlinski et
al., 2005). Furthermore, Homer1 KO mice exhibit sensorimotor impairments, which can
be attenuated with the typical antipsychotic, haloperidol (Szumlinski et al., 2005). Whilst
these studies implicate Homer1’s involvement in behavioural impairments associated
with neuropsychiatric disorders, they do not discriminate which Homer1 isoforms may
underlie these behaviours. However, the use of adeno-associated virus (AAV) to induce
expression of specific Homer1 isoforms in Homer1 KO models has provided a tool to
elucidate this issue. Homer1 KO mice infused with the AAV Homer1 long isoform in the
prefrontal cortex, reversed stress related learning impairments, observed by deletion of
Homer1. In regards to the short Homer1 isoform, upregulation of Homer1a has been
observed following psychotomimetic drug exposure, the introduction to novel
environments, fear conditioning and avoidance learning tasks (Cochran et al., 2002;
Fujiyama et al., 2003; Igaz et al., 2004; Vazdarjanova et al., 2002). Collectively, this
suggests that the short form, Homer1a, may be involved in adaptive behaviours
following stress. Furthermore, AAV infusion of Homer1c, but not Homer1a, in the
prefrontal cortex of Homer1 KO mice, reversed working memory and sensorimotor
deficits (Lominac et al., 2005). In addition, Homer1 KO mice exhibit increased
extracellular glutamate in the prefrontal cortex, which was reversed by AAV infusion of
Homer1c (Lominac et al., 2005). This suggests that Homer1c plays an integral role in
cognitive and sensorimotor processing, which may be associated with frontal cortical
glutamate levels. In addition, it appears Homer1c may also play a role in hippocampal
dependent memory, as AAV infusion of Homer1c in the hippocampus has demonstrated
to restore spatial memory and LTP deficits in Homer1 KO mice (Gerstein et al., 2012).
Moreover, this effect was abolished by mGluR5, but not mGluR1, antagonism indicating
Homer1c facilitates synaptic plasticity effects via its interaction with mGluR5 (Gerstein
et al., 2012). Collectively, these findings in rodents highlight the distinct role Homer1

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isoforms play in behaviours associated with neuropsychiatric disorders and therefore
may play a role in the pathology of these disorders.

A genetic study examining HOMER1, provided the first evidence implicating Homer1 in
schizophrenia. Norton et al., (2003) identified several single nucleotide polymorphisms
were associated with schizophrenia and other neuropsychiatric disorders. In addition,
Spellmann and colleagues (2011) identified HOMER1 polymorphisms are associated
with symptom severity and antipsychotic response. Furthermore, antipsychotics have
demonstrated to regulate HOMER1 gene expression in rodent models (discussed later
in Section 1.7). Despite these findings, there has been limited investigation of Homer1
in postmortem cohorts. Engmann et al., (2011) reported reduced Homer protein
expression in the PFC and hippocampus of a postmortem schizophrenia cohort, however
the antibody employed in this study was not specific for any Homer isoform. We recently
reported an increase in Homer1a and a decrease in Homer1b/c protein expression in
the CA1 of a schizophrenia postmortem cohort, in which we also showed concomitant
mGluR1 and mGluR5 alterations (Matosin et al., 2015c, 2016b). Whether the alterations
in Homer1 in schizophrenia extends to other brain regions is yet to be investigated.

1.6.4 Norbin
While constitutive Norbin KO is lethal (Mochizuki et al., 2003), conditional Norbin KO
mice have previously been generated from the Greengard lab (Wang et al., 2009). In
these mice Norbin protein is deleted specifically from the postnatal forebrain, leaving
mid- and hind-brain mGluR5 intact. Conditional Norbin KO mice exhibit reduced cell
surface expression of mGluR5, however overall expression appears unaltered, indicating
Norbin is responsible for trafficking mGluR5 to the cell surface (Wang et al., 2009).
Norbin KO mice display many behaviours reminiscent of neuropsychiatric models,
including pre-pulse inhibition abnormalities and increased hyperlocomotive response to
MK-801 (Wang et al., 2009). Furthermore, they also demonstrate depressive like
behaviours, with reports of reduced mobility times in forced swim and tail suspension
tests (Wang et al., 2015). Further investigation also show these mice exhibit many
neurochemical abnormalities associated with schizophrenia and depression. Norbin KO
mice exhibit reduced DHPG (group 1 mGluR agonist)-induced LTP and LTD in

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hippocampal Schaffer collateral-CA1 synapses, signifying synaptic plasticity deficits in
these mice (Wang et al., 2009). In addition, Norbin ablation reduces adult neurogenesis
in the subgranular zone, reducing the proliferation of neural stem cells. Further analysis
showed that these impairments in adult neurogenesis were through non-autonomous
effects as Norbin expression was extremely low in neural stem and precursor cells (Wang
et al., 2015). Although our knowledge about Norbin and its role in neuropsychiatric
disorders is still within its infancy, Norbin provides a novel candidate molecule of
interest, which warrants further investigation.

The work investigating pathological changes to Norbin in psychiatric postmortem

cohorts is still in its infancy. However, Mudge and colleagues reported reduced Norbin
mRNA levels in the cerebellar cortex of schizophrenia subjects (Mudge et al., 2008).
Furthermore, we recently reported increased Norbin protein levels in the CA1 region of
the hippocampus (Matosin et al., 2015c) and reductions in the dorsolateral prefrontal
cortex (BA46) of schizophrenia subjects (Matosin et al., 2015a), indicating unique brain
region effects. Contrary to this, Martins-de-Souza et al., (2012) showed a modest
increase of Norbin protein levels within the dorsolateral prefrontal cortex (BA9) of major
depression subjects with psychosis compared to those without psychosis. This evidence
implicating Norbin in the pathology of schizophrenia and depression, may consequently
contribute to changes in localisation and signalling of group 1 mGluRs.

1.7 Effect of Current Antipsychotic Drugs on Group 1 mGluR

Antipsychotic drugs are widely used in the treatment of schizophrenia, particularly the
positive symptoms, with all current antipsychotics sharing a strong affinity for the
dopamine D2 receptor (Kapur and Mamo, 2003). First generation antipsychotics such as
haloperidol are primarily D2 receptor antagonists, whilst second generation
antipsychotics such as clozapine, olanzapine and sertindole have additional affinity for
other neurotransmitter systems such as the serotonergic, muscarinic and histaminergic
systems (Mauri et al., 2014). First and second generation antipsychotics primarily treat
only positive symptoms. Furthermore, chronic use of these compounds is accompanied
by an array of side effects such as extrapyramidal symptoms, agranulocytosis, weight

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gain, type 2 diabetes mellitus and hypercholesterolaemia (Henderson et al., 2000). More
recently, a third generation of antipsychotics was developed, most notably aripiprazole,
which has shown favourable efficacy for the treatment of schizophrenia and reduced
side-effects (Di Sciascio and Riva, 2015). Whilst aripiprazole is a D2 receptor partial
agonist (Shapiro et al., 2003), the mechanisms underlying its therapeutic profile appear
complex and largely unknown. There is particular benefit in attempting to understand
the neurochemical changes antipsychotics may induce for several reasons: 1. To offer a
comparison against findings from postmortem studies and discriminate between
pathological changes and changes which could be induced by antipsychotics; 2. Identify
antipsychotics that may target certain neurotransmitter systems that could profit a sub
group of patients; 3. Identify potential “off target” side effector systems and 4. Identify
novel therapeutic targets.

Despite the fact that current antipsychotics do not have affinity for the range of
glutamatergic receptors, evidence indicates antipsychotics indirectly modulate the
glutamatergic system. Several studies demonstrate antipsychotic drugs, including
haloperidol and aripiprazole, can alter the ligand binding, mRNA and protein expression
of glutamatergic ionotropic receptors, in particular AMPA and NMDA receptors, within
the nucleus accumbens (Fitzgerald et al., 1995; Healy and Meador-Woodruff, 1997; Pan
et al., 2016; Schmitt et al., 2003; Spurney et al., 1999). Studies investigating the effect
of antipsychotics on glutamatergic receptors have largely focused on ionotropic
receptors, limited studies have investigated the influence of antipsychotics on group 1
mGluRs. Volk et al., (2010) reported chronic (17-27 months) treatment with either
haloperidol (2.0-4.0 mg/day) or olanzapine (1.1-2.2 mg/day) had no effect on mGluR1a
mRNA in the prefrontal cortex of Macaque monkeys. Similarly, a rodent study did not
observe a change in mGluR1 or mGluR5 transcripts in the frontal cortex, striatum or
hippocampus following antipsychotic treatment (3 weeks; haloperidol 1 mg/kg;
clozapine 10 mg/kg and olanzapine 2 mg/kg) (Tascedda et al., 2001). This is in line with
studies conducted in our laboratory, which have also reported unchanged mGluR5
protein expression within the prefrontal cortex and hippocampus of rodents chronically
treated with haloperidol (0.3 mg/kg dose; 8, 16 and 36 days) or olanzapine (3 mg/kg; 8,

Jeremy Lum 53
16 and 36 days duration) (Matosin et al., 2015a, 2015c). Furthermore, we did not detect
a change in [3H] MPEP binding to mGluR5 in the rodent prefrontal cortex, hippocampus
or striatum following haloperidol or olanzapine treatment (Matosin et al., 2013). Whilst
the aforementioned studies examined the entire regional structures, it appears there
may be sub-regional differences, particularly within the striatum. Iasevoli et al., (2010)
reported increased mGluR5 mRNA expression in the dorsal striatum (putamen)
following chronic treatment with haloperidol or sertindole, however, this did not extend
to the ventral striatum (nucleus accumbens). There appears to be little evidence to date
suggesting group 1 mGluR mRNA or protein expression is influenced following
administration of first and second generation antipsychotics. However, protein
expression studies have only examined the monomeric form of mGluR5 and not the
dimeric form, which is now known to be the functional form required for agonist-
activation and downstream signalling (El Moustaine et al., 2012). Furthermore, group 1
mGluR protein expression has yet to be examined following administration with a third
generation antipsychotic, such as aripiprazole, which has superior long-term efficacy
and more favourable adverse effect profile, than haloperidol (Kasper et al., 2003; Stip
and Tourjman, 2010). Understanding the neurochemical changes associated with
advantageous treatments such as aripiprazole, particularly changes core its therapeutic
efficacy can improve future drug design.

Studies investigating proteins that endogenously regulate group 1 mGluRs, particularly

Homer1, have shown to be influenced by antipsychotic treatment. A large body of work
from de Bartolomeis and colleagues has revealed the influence antipsychotics can have
on Homer1 expression, in particular the short form, Homer1a (Ambesi-Impiombato et
al., 2007a; de Bartolomeis et al., 2002a; Iasevoli et al., 2010b, 2011a; Tomasetti et al.,
2007). Interestingly, evidence from this work indicates antipsychotic D2 receptor
blockade is positively associated with Homer1a expression, specifically in the striatum
(Ambesi-Impiombato et al., 2007; de Bartolomeis et al., 2016). de Bartolomeis and
colleagues (2015) reported acute treatment with the typical antipsychotic, haloperidol,
induces a gradual dose-dependent increase of Homer1a mRNA expression in the
striatum. Furthermore, acute treatment with 2nd and 3rd generation antipsychotics

Jeremy Lum 54
olanzapine, clozapine, asenapine, ziprasidone and aripiprazole, have also shown to
increase Homer1a mRNA in the striatum (de Bartolomeis et al., 2002, 2015b; Iasevoli et
al., 2010b, 2010a; Polese et al., 2002; Tomasetti et al., 2007). However, sertindole, a
second generation antipsychotic did not show an effect on Homer1a expression in the
striatum, which may highlight that various antipsychotics preferentially induce Homer1a
expression (Iasevoli et al., 2010b). In addition, antipsychotics may induce Homer1a
expression in a brain region specific manner. In fact, acute treatment with haloperidol,
olanzapine or clozapine has shown to have no effect on hippocampal Homer1a
expression, however has shown to increase Homer1a expression in cortical and striatal
regions (Iasevoli et al., 2009, 2010b). The striatum, in particular the nucleus accumbens,
is a region of great interest not only because it is thought to be the “action” site of
antipsychotics (due to its high concentration of D2 receptors), but it also contains an
abundance of glutamatergic terminals, where Homer1 is localised. Collectively, studies
suggest acute treatment with 1st, 2nd and even 3rd generation antipsychotic drugs can
increase Homer1a mRNA in the nucleus accumbens, however, Homer1b/c levels are
largely reported to be unchanged. Considering the role of Homer1a, this suggests that
acute antipsychotic treatment may alter mGluR1/5 signalling in the nucleus accumbens,
uncoupling its attachment to IP3 and shifting its signalling towards G-protein
independent pathways. However, it should be noted that these previous studies have
focused on mRNA levels, which may not reflect protein levels (Greenbaum et al., 2003).
Future studies should examine the protein expression of these endogenous modulators
and their interaction with group 1 mGluRs to gain a deeper insight into their potential
therapeutic significance.

While a growing body of evidence indicates acute antipsychotic treatment influences

Homer1 expression, patients in the clinic undergo chronic treatment, therefore,
appropriately modelling and understanding the chronic effect of these antipsychotics
may provide more relevant information to the clinical situation. Furthermore, studies
have reported differential mRNA and protein responses to short- and long-term
antipsychotic administration (Deng et al., 2015). Such studies can identify if acute effects
are preserved or if chronic treatment may cause neuro-adaptations that are key to their

Jeremy Lum 55
efficacy. Furthermore, considering Homer1 plays a key role in synaptic plasticity, insights
into the long-term effect of antipsychotics may identify the therapeutic mechanism of
antipsychotics. There have been several studies investigating this in regards to Homer1
within the nucleus accumbens. However, reports have been inconsistent reporting no
change and increases of Homer1a and Homer1b/c mRNA transcripts within identical
models (Ambesi-Impiombato et al., 2007; Iasevoli et al., 2010b, 2011; Tomasetti et al.,
2007). Whilst it is unclear why there are inconsistencies within identical models, further
studies examining this area may highlight the role Homer1 plays in antipsychotic-
induced synaptic plasticity.

When modelling the antipsychotic drug exposure in rodent models, several

considerations should be noted to ensure clinical relevance. It is difficult to make direct
translations between human and rodents based on body weight as drug absorption and
metabolism differ between species. However, D2 receptor occupancy is a widely
accepted parameter to translate dosage (Kapur, 1998; Kapur et al., 2003). In regards to
drug delivery, several drug delivery methods have been used in previous studies,
including mini-pumps, intraperitoneal or subcutaneous injection, oral gavage and oral
self-administration via drinking water, however these methods have limitations that
must be considered. As such, studies thus far investigating the effect of chronic
antipsychotic treatment on Homer1 expression have employed a daily intraperitoneal
injection paradigm (Ambesi-Impiombato et al., 2007; de Bartolomeis et al., 2002, 2013,
2015b; Iasevoli et al., 2010a, 2011; Spellmann et al., 2011; Tomasetti et al., 2007).
Injections and oral gavage require regular handling and place stress on the rodent, which
may place limitations on results, in particular on Homer1, which is known to be
influenced by stress (Shui et al., 2015; Wagner et al., 2015). Furthermore, the half-life of
antipsychotics is 4-6 times faster than humans, therefore, rodents must be dosed higher
and/or more frequently to achieve similar D2 receptor occupancy levels to humans
(Kapur et al., 2003). Whilst delivery via drinking water may overcome these problems,
water intake may fluctuate, consequently causing maintenance of a steady dose
problematic. Furthermore, several antipsychotics are not readily water-soluble.
Similarly, antipsychotic delivery through mini-pumps can lead to degradation of the

Jeremy Lum 56
compound. However, a number of studies have employed oral self-administration
through a delivery of the antipsychotic encased in a pleasant tasting treat such as peanut
butter or cookie-dough (Han et al., 2008; Huang-Brown and Guhad, 2002). Not only does
oral administration better replicate the clinical scenario, it avoids invasive surgeries,
unnecessary handling and stress associated with injections and/or oral gavage.
Furthermore, antipsychotics can be given at numerous times throughout the day to keep
D2 receptor occupancy levels stable. Prospective studies investigating the effects of
chronic antipsychotic treatment on Homer1 expression may benefit from using such a
treatment paradigm.

1.8 mGluR5 positive allosteric modulation as a therapeutic target for

schizophrenia
There is a pressing need to develop novel therapeutic approaches for treating
schizophrenia that not only avoid the debilitating side effects but also, effectively treat
all symptoms domains of the disorder. mGluR5 was originally identified as a therapeutic
target for schizophrenia due to its ability to modulate NMDA receptor deficits (Kinney
et al., 2005), which are thought to be associated with schizophrenia, at least in a subset
of schizophrenia patients. However, due to the distribution and dynamic signalling
pathways of mGluR5 (for review see Matosin and Newell, 2013), several other potential
therapeutic mechanisms are thought to be possible (Rook et al., 2015). Pharmacological
stimulation of mGluR5 has shown promising potential to improve cognitive behaviours
(Ayala et al., 2009; Uslaner et al., 2009; Clifton et al., 2013). While mGluR5 is considered
a novel therapeutic target, agonist stimulation of mGluR5 in rodents has been reported
to cause seizures and rapid receptor desensitisation (Wong et al., 2005). However, in
the last decade a new class of mGluR5 stimulators, in the form of positive allosteric
modulator (PAM) compounds, have been synthesised to specifically target mGluR5
(O’Brien et al., 2003).

mGluR5 PAMs are a class of compound which bind within the transmembrane region of
mGluR5 and cause a conformational change to the receptor, generally without exerting
an intracellular signalling response on their own, in contrast to a typical orthosteric

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agonist (Vinson and Conn, 2012). However, when a PAM is co-bound with an mGluR5
endogenous orthosteric ligand (i.e. glutamate), the activity of mGluR5 is heightened and
the PAM further potentiates downstream signalling pathways (Kinney et al., 2005). Since
the first development of mGluR5 PAMs over a decade ago, there has been a surge in the
number of developed and tested compounds. mGluR5 PAMs which have been examined
in vivo include ADX-47273 (developed by Addex Therapeutics), CDPPB (3-Cyano-N-(1,3-
diphenyl-1H-pyrazol-5-yl)benzamide) (developed by Merck) and VU-analogues
(developed by the Vanderbilt Centre for Neuroscience Drug Discovery).

mGluR5 PAMs have provided a new therapeutic strategy to attenuate several

schizophrenia-like behaviours in animal models. A variety of mGluR5 PAMs have shown
success in reversing a wide range of positive- and negative-like behaviours induced via
NMDA receptor antagonist or dopaminergic agonist agents, including hyperlocomotion,
pre-pulse inhibition and sucrose preference deficits (for detailed review see Matosin and
Newell, 2013). However, one of the most promising features of mGluR5 PAMs is their
ability to offset social, spatial and working memory deficits, a property not associated
with current antipsychotics. mGluR5 PAMs have demonstrated efficacy to attenuate
cognitive deficits including in the Morris water maze, social novelty discrimination, set
shifting performance, novel object recognition and conditioned avoidance tasks in
various animal models of schizophrenia (Ayala et al., 2009; Balschun et al., 2006; Chan
et al., 2008; Darrah et al., 2008; Horio et al., 2013; Liu et al., 2008; Stefani and
Moghaddam, 2010; Uslaner et al., 2009). Collectively, the preclinical evidence for
mGluR5 PAMs is promising for treating a wide range of behaviours associated with
schizophrenia. Recently mGluR5 PAMs have been further investigated to treat other
neuropsychiatric and neurological disorders as well. In particular, the mGluR5 PAM,
CDPPB has demonstrated potential in the treatment of autism (Won et al., 2012), Rett
syndrome (Gogliotti et al., 2016), post-traumatic stress disorder (Sethna and Wang,
2014), Huntington’s disease (Doria et al., 2013), ethanol and cocaine seeking behaviour
(Cleva et al., 2011). Whilst these studies investigating CDPPB are in early preclinical
stages, the evidence is favourable and encourages further investigation.

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1.8.1 Therapeutic potential of CDPPB
CDPPB has been the most extensively investigated mGluR5 PAM to date, demonstrating
antipsychotic and pro-cognitive effects in schizophrenia rodent models. Kinney et al,
(2005) showed CDPPB has low oral availability (F=2.9%) when delivered in 1%
methycellulose. However, intravenous injection CDPPB (2 mg/kg; dimethyl sulfoxide
(DMSO)) in Sprague-Dawley rats has a plasma half-life of 4.4 hrs, with a volume
distribution of 3.5 l/kg and clearance of 25.1 ml/min/kg (Kinney 2005). Furthermore,
CDPPB is brain penetrable with a brain/plasma ratio of 1.7 (Kinney 2005), indicating it is
a suitable compound for in vivo investigation.

Similar to many of the other PAMs, CDPPB competes with binding of the MPEP binding
site (Kinney et al., 2005). CDPPB does not increase agonist binding affinity, nor does
glutamate affect CDPPB binding at the MPEP binding site (Kinney et al., 2005). Rather,
CDPPB exerts its effect through increasing the functional potency of the mGluR5
orthosteric agonist. One unexpected property of CDPPB is its ability to activate mGluR5
in the absence of an agonist (Kinney et al., 2005; Noetzel et al., 2012). Therefore, CDPPB
has been termed an ago-PAM, rather than a “pure” PAM. However, it has been
demonstrated in HEK293 cells that the agonist properties of ago-PAMs are dependent
on mGluR5 expression levels, with CDPPB showing agonist activity in the presence of
high levels of mGluR5. However all ago-PAMs, including CDPPB, have shown no agonist
activity in primary cells, including cortical astrocytes and cultured neurons from the
subthalamic nucleus; this suggests mGluR5 PAMs, such as CDPPB, may exhibit agonist
activity in recombinant cell lines, however does not reflect or predict agonist activity in
native systems (Noetzel et al., 2012).

Acute CDPPB treatment has shown antipsychotic-effects in attenuating NMDA receptor

antagonist- and amphetamine-induced hyperlocomotion and pre-pulse inhibition
deficits (Fowler et al., 2011, 2013; Kinney et al., 2005a). In addition, CDPPB was shown
to attenuate MK-801-induced anhedonia and immobility time in the sucrose preference
and forced swim tests, behaviours used to assess negative-like symptoms (Vardigan et
al., 2010; Wierońska et al., 2015). However, the most promising aspect of mGluR5 PAMs,

Jeremy Lum 59
including CDPPB are their pro-cognitive effects. CDPPB was shown to potentiate both
LTP and LTD within the Schaffer collateral-CA1 synapse of hippocampal slices (Ayala et
al., 2009). This is further supported by CDPPB’s ability to enhance rodent performance
in the Morris water maze (Ayala et al., 2009). Furthermore, acute CDPPB administration
has demonstrated to attenuate NMDA receptor antagonist-induced deficits in novel
object recognition and cognitive flexibility tasks (Lacrosse et al., 2015; Stefani and
Moghaddam, 2010; Uslaner et al., 2009).

Collectively, studies investigating acute CDPPB treatment report great efficacy in

attenuating schizophrenia-like behaviours (Table 1.3). However, in an attempt to mimic
a more clinically relevant situation, studies have focused on investigating the effects of
chronic CDPPB administration. The first of these studies demonstrated seven-day CDPPB
treatment (30 mg/kg; i.p) showed the ability to reduce amphetamine-induced
hyperlocomotion in adult rats (Parmentier-Batteur et al., 2012). This effect was to the
same degree as rodents treated acutely with CDPPB, indicating tolerance is not
developed following chronic CDPPB treatment, at least in this paradigm. In addition, this
study also included a withdrawal group, which received CDPPB for six days, yet did not
receive CDPPB treatment on the day of the amphetamine challenge. Rodents in the
withdrawal group did not display any efficacy in attenuating amphetamine-induced
hyperlocomotion, suggesting the effects of CDPPB are reversible following even a brief
washout period (Parmentier-Batteur et al., 2012). Horio and colleagues (Horio et al.,
2013) examined both the acute and chronic effects of CDPPB (10 mg/kg; i.p) in
ameliorating PCP-induced (10 mg/kg for consecutive 10 days) cognitive deficits in adult
mice. Chronic (14 day), but not acute, CDPPB treatment was able to attenuate PCP-
induced deficits in novel object recognition (Horio et al., 2013). Subsequently, Clifton
and colleagues brought attention to the idea that early mGluR5 PAM intervention may
be able to prevent schizophrenia-like behaviours. They were able to demonstrate
chronic CDPPB (10 mg/kg; i.p; 12 days) treatment at adolescence (PN35-46) was able to
attenuate social cognitive deficits induced in the perinatal PCP model of schizophrenia
(Clifton et al., 2013). Furthermore, these effects were observed in adulthood, six weeks
after withdrawal from treatment. This supports the possibility that early intervention,

Jeremy Lum 60
targeting mGluR5, may be a future therapeutic avenue, however this is the only study
to investigate adolescent mGluR5 PAM treatment and therefore further investigation is
required.

Despite CDPPB’s promising ability to treat schizophrenia-like symptoms in rodent

models, there is still little known in regards to CDPPB’s effect at a molecular, cellular and
circuitry level. The major attraction of mGluR5 PAMs, including CDPPB, is their ability to
counteract NMDA receptor dysfunction. In line with this, CDPPB (3 and 10 mg/kg; i.p)
was demonstrated to attenuate spontaneous activity of medial prefrontal cortex
neurons, induced via the NMDA receptor antagonist, MK-801, supporting the potential
of CDPPB to treat NMDA receptor deficits (Lecourtier et al., 2007). Furthermore, Chen
et al., (2011) demonstrated CDPPB was able to restore the reduced field potentials
induced via the NMDA receptor antagonists, ketamine (non-competitive) and D-APV
(competitive), in hippocampal slices. This was mediated via CDPPB’s ability to modulate
the PKC pathway, as application with the PKC inhibitor, chelerythrine chloride, blocked
this effect. PKC activation was shown to phosphorylate NMDA receptors as well as
activate CAMKII and CREB pathways, which are downstream of the PKC pathway
(Benquet et al., 2002; Jia et al., 1998). Uslaner et al., (2009) demonstrated acute CDPPB
treatment (3, 10 and 30 mg/kg; i.p) increased synaptic NR1 (ser896) and NR2B (1303)
phosphorylation in the frontal cortex and hippocampus and increase phosphorylation of
the AMPA GluA1 subunit (ser845) at various doses. Whilst these results support a role
of CDPPB to upregulate NMDA and AMPA receptor activity, the study showed dose- and
brain region- dependent responses. In the frontal cortex, phosphorylation of NR1, NR2B
and GluA1 showed an inverted U-dose dependent effect, whilst in the hippocampus a
linear dose-effect was observed, indicating CDPPB may exert brain-region specific
differences. In support of these brain region specific differences, Parmentier-Batteur et
al., (2012) reported that acute administration of CDPPB (30 mg/kg) in adult rats caused
increased phosphorylation of NR1 and NR2B (ser1303) in the frontal cortex and
striatum; this effect was still observed in the striatum following 7-day CDPPB treatment,
but was absent in the frontal cortex. Whilst mGluR5 PAMs have shown promising
behavioural potential, knowledge of their neurochemical effects is still limited.

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Furthermore, studies examining the neurochemical effects of CDPPB have been
performed in adult rats. In light of the notion that adolescent CDPPB treatment may be
a potential therapeutic strategy, it is imperative to investigate if adolescent CDPPB
treatment causes similar neurochemical changes, as reported in adult rats and if these
changes are long-term. A deeper understanding of the neurochemical effect of CDPPB
may enhance the development of not only CDPPB, but also mGluR5 as a treatment for
schizophrenia and potentially other neuropsychiatric disorders.

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Table 1.3: Summary of preclinical studies assessing the behavioural and neurochemical efficacy of CDPPB in rodents.
Rodent age Model/CDPPB dose Reported results Reference
Behavioural Findings
Adult Sprague- Open field test: Kinney et al, 2005
Dawley rats  CDPPB (1, 3 and 30 mg/kg s.c.) 20 mins prior to amphetamine (1  CDPPB dose-dependently decreased amphetamine-induced hyperlocomotion
mg/kg s.c). activity.

Pre-pulse inhibition:
 CDPPB (3, 10 and 30 mg/kg s.c.) 20 mins prior to amphetamine (2  CDPPB (10 and 30 mg/kg) attenuated amphetamine-induced pre-pulse
mg/kg s.c.). inhibition deficits.

Adult male Morris water maze: Alaya et al., 2009

Sprague-Dawley  CDPPB (10 mg/kg) 20 mins prior to testing during seven-day training.  CDPPB enhanced performance in Morris water maze.
rats
Adult male Wistar Novel Object Recognition: Uslaner et al, 2009
Hannover  CDPPB (0, 10 and 30 mg/kg; i.p) administration in unimpaired rats.  CDPPB (10mg/kg) enhanced novel object recognition performance.

 CDPPB (0, 10 and 30 mg/kg; i.p) immediately prior to MK-801 (0.3  CDPPB (3mg/kg) attenuated MK-801-induced recognition memory deficits.
mg/kg; i.p) .

Adult male Wistar- Sucrose preference test: Vardigan et al, 2010

Hannover rats  CDPPB (3 mg/kg i.p), 30mins prior to MK-801 treatment (0.3mg/kg).  CDPPB treatment attenuated MK-801 induced anhedonic behaviour similar to
clozapine and D-serine.
Adult male Set shifting task: Stefani and
Sprague-Dawley  CDPPB (10 or 30 mg/kg; i.p), 20 mins following MK-801 (0.1 mg/ml)  CDPPB (10 and 30 mg/kg) attenuated MK-801 induced set shifting deficits. Moghaddam 2010
rats treatment.
Adult male Inhibitory avoidance, open field test and taste aversion tests: Inhibitory Avoidance: Fowler et al. 2011
Sprague-Dawley  CDPPB (3 and 10mg/kg, i.p) simultaneously with MK-801 (0.2mg/kg)  CDPPB (3 mg/kg) treatment attenuated MK-801-induced latencies.
rats administration.
Open Field test:
 CDPPB (3 mg/kg) attenuated MK-801-induced hyperlocomotion.

Conditioned taste aversion:

 CDPPB (3 mg/kg) attenuated MK-801-induced disruption in condition taste
aversion..

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Male ICR mice (6 Morris water maze: Horio et al., 2012
weeks old) PCP (10 mg/kg) from experimental days 1-5 and 8-12
 Acute CDPPB (10mg/kg; i.p) 3 days following last PCP injection  CDPPB did not attenuate PCP-induced recognition memory deficits.
(experimental day 15).

 Chronic CDPPB (1 or 10mg/kg; i.p) 3 days following last PCP injection  CDPPB (10mg/kg) attenuated PCP-induced recognition memory deficits.
(experimental day 15) for 14 consecutive days.
Adult Wistar rats Amphetamine-induced hyperlocomotion: Parmentier-Batteur
 Acute or daily CDPPB (30 mg/kg; i.p) treatment for either 6 or 7 days  Acute and 7-day CDPPB treatment group attenuated amphetamine-induced et al., 2012
prior to amphetamine (0.75mg/kg) challenge. hyperlocomotion.
Male Wistar rats Social novelty discrimination: Clifton et al., 2013
Parametric deficits protocol:
 CDPPB (0.16, 2.5, 10 and 40 mg/kg; i.p).  CDPPB (2.5 and 10 mg/kg) improved social novelty discrimination performance.

Parametric deficits protocol:

 CDPPB was infused bilaterally in the frontal cortex (0.63, 2.5, 10  CDPPB (2.5 ug/side) infusion within frontal cortex improved ocial novelty
ug/side) or striatum (2.5 ug/side). discrimination performance. No effect was observed in striatum.

Pharmacological deficit protocol:

 CDPPB (10 mg/kg; i.p) 45 mins following MK-801 (0.08 mg/kg; s.c).  CDPPB attenuated MK-801 induced social novel discrimination deficits.

Perinatal PCP model: PCP (10 mg/kg; s.c) injections on PN7, 9 and 11.
 Adolescent (PN35-46) CDPPB (10 mg/kg/day; s.c)  Adolescent CDPPB prevented perinatal PCP-induced social novelty
 Adult (PN70-81) CDPPB (10 mg/kg/day; s.c) discrimination deficits when tested at 8 weeks and 13 weeks.
 Adult CDPPB treatment had no effect on perinatal PCP-induced social novelty
discrimination deficits when tested at 13 weeks.
Adult male C57B1/6 Barnes maze task: Fowler et al., 2013
mice  CDPPB (10 or 30 mg/kg; i.p) 20 mins prior to reversal training for 3  CDPPB (30 mg/kg) enhanced reversal learning performance.
days.

T-maze task:
Adult male Sprague  CDPPB (10 or 30 mg/kg; s.c) 20 mins prior training for 5 days.  CDPPB (30 mg/kg) enhanced T-maze acquisition performance.
Dawley rats
Adult male Set shifting task: LaCrrosse et al.,
Sprague-Dawley  CDPPB (20 mg/kg) 30mins prior to MK-801 (0.06 mg/kg).  CDPPB treatment prevented MK-801 induced set-shifting deficits. 2014
rats

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Adult male Albino Forced swim test: Wieronska et al.,
Swiss mice  MK-801 (0.4 mg/kg, i.p) for 13 days, with a 1-day washout period prior  CDPPB (0.5, 1 and 2mg/kg) reduced chronic MK-80-induced immobility time. 2015
to forced swim test. CDPPB (0.1, 0.5, 1 and 2 mg/kg) was administered
30 mins before forced swim.

DOI-induced head twitches:

 CDPPB (1, 5 and 10 mg/kg) was administered was 30 mins prior to DOI  CDPPB (1, 5 and 10 mg/kg) inhibited DOI induced head twitches.
(2.5 mg/kg; i.p).

Haloperidol induced catalepsy:

 CDPPB (0.25, 0.5, 1 and 2 mg/kg) was administered was 30 mins prior  CDPPB (0.25, 0.5, 1 and 2 mg/kg) dose-dependently inhibited haloperidol
to haloperidol (0.1 mg/kg) induced catalepsy.

Adult male Wistar Novel object recognition task:

rats  CDPPB (1, 2 and 5 mg/kg) was administered 30 mins prior to MK-801  CDPPB (2 and 5 mg/kg) attenuated MK-801-induced novel object recognition
administration. deficits.
Adult male Long- Paired associated learning: Lins and Howland.,
Evans rats  CDPPB (3, 10 and 30 mg/kg; i.p) was administered 5 mins prior to MK-  CDPPB did not attenuate MK-801-induced learning deficits. 2016
801 (0.15 mg/kg; i.p) administration.

Neurochemical findings
Adult male Electrophysiology:  CDPPB blocked MK-801-induced neuronal firing activity deficits in medial Lecourtier et al.,
Sprague-Dawley  CDPPB (10 mg/kg; i.p) 30 mins prior to MK-801 (0.1 mg/kg; i.p) prefrontal cortex. 2007
rats administration.
Microdialysis:
 CDPPB (10 mg/kg; i.p) 20 mins prior to MK-801 (0.1mg/kg; i.p)  CDPPB treatment did not reverse MK-801 induced dopamine release in the
administration. medial prefrontal cortex and nucleus accumbens.
Adult male Wistar Unimpaired rats administered CDPPB (0, 10 and 30 mg/kg; i.p) and  CDPPB (10 and 30mg/kg) increased synaptic expression and phosphorylation of Uslaner et al, 2009
Hannover euthanised 1 hr after CDPPB treatment. pNR1 (ser896) and pNR2B (ser1303) in the hippocampus.
 CDPPB (3 and 10mg/kg) increased synaptic pNR1 and pNR2B in frontal cortex.
 CDPPB (3,10 and 30 mg/kg) increased pαCAMK11 (Thr286) expression in the
hippocampus.
 CDPPB (10mg/kg) increased pCREB in hippocampus and PFC.
 CDPPB (3 and 10 mg/kg) increased pGluA1 in hippocampus and PFC.
Adult Wistar rats Acute or daily CDPPB (30 mg/kg; i.p) treatment for either 6 or 7 days  CDPPB treatment for 7-days reduced [3H] bMax and kD in PFC, but not striatum Parmentier-Batteur
prior to amphetamine (0.75mg/kg) challenge.  Acute and 7-day CDPPB treatment increased pNR1 (ser896), pNR2B (ser1303) et al., 2012
in striatum and frontal cortex.
Abbreviation: s.c, subcutaneous; i.p, intraperitoneal; PN, postnatal day; PCP, phencyclidine.

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1.9 Summary
Disturbances to the glutamatergic system are widely believed to contribute to the
pathogenesis of schizophrenia. In particular, genetic and environmental evidence
indicates neurodevelopmental disruption to the NMDA glutamate receptor may
underlie the disorder. In line with this, pharmacological blockade of the NMDA receptor,
with antagonists such as PCP, during development produces behaviours and
neurochemical alterations analogous to schizophrenia. Therefore, this paradigm
provides a model of schizophrenia, to examine the underlying pathogenesis and
investigate potential new treatments.

The NMDA receptor has been shown to share a physical and functional interaction with
group 1 mGluRs (mGluR1 and mGluR5). mGluR1 and mGluR5 are 7-transmembrane
spanning glutamate receptors that positively couple to Gq/11 and form individual
homodimers, which is vital for their downstream signalling. Furthermore, they are
important regulators of glutamatergic transmission, brain development and play a role
in a wide variety of behaviours, most notably cognitive function, processes implicated in
the pathology of schizophrenia. Moreover, genetic deletion of mGluR1 or mGluR5 in
rodent models induces schizophrenia-like behaviours. Therefore, it is conceivable group
1 mGluRs may contribute to the pathogenesis of schizophrenia.

Recent postmortem studies reported altered expression of group 1 mGluRs in the

prefrontal cortex and hippocampus, regions that send glutamatergic projections to the
nucleus accumbens. Whilst the nucleus accumbens has previously been associated with
dopaminergic alterations, recent investigation in low powered postmortem cohorts
indicate the nucleus accumbens exhibits glutamatergic abnormalities in schizophrenia.
Furthermore, a large body of work in rodent models indicates that current
antipsychotics, which primarily target the dopaminergic system, alter glutamatergic
tone within the nucleus accumbens. However, further research is required to
characterise the glutamatergic system in the nucleus accumbens, in regards to the
pathology and current pharmacological treatment of schizophrenia.

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In addition to exploring group 1 mGluRs in the pathology of schizophrenia, recent drug
discovery efforts have focused on these receptors as novel targets as an avenue to
upregulate NMDA receptor deficits associated with schizophrenia. In particular, mGluR5
PAMs such as CDPPB, have been shown to potentiate NMDA receptor currents and
attenuate schizophrenia-like behaviours, in particularly cognitive deficits, in rodent
models. Furthermore, CDPPB administration has also demonstrated potential for the
treatment of other neurological and neuropsychiatric disorders such as autism, Rett
syndrome, post-traumatic stress disorder, Huntington’s disease and addiction.
However, there is still much unknown as to the molecular effects of CDPPB and other
mGluR5 PAMs. Further understanding the underlying mechanisms, which may mediate
their therapeutic efficacy, will assist in the development of these compounds for clinical
suitability.

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1.10 Aims and Hypotheses

1.10.1 General Aim:

To investigate group 1 mGluRs in the pathology and treatment of schizophrenia, in
addition to their therapeutic potential.

1.10.2 Specific Aims:

1. To characterise the protein expression of group 1 mGluR and their endogenous
regulators in the nucleus accumbens (a region associated with the positive
symptoms of schizophrenia and regarded as critical for the therapeutic action of
current antipsychotics) of a postmortem schizophrenia cohort.

2. To determine the short- and long-term effect of commonly used antipsychotic

treatments on the protein expression of group 1 mGluRs and their endogenous
regulators, in the nucleus accumbens of a rodent model.

3. To examine the neurodevelopmental profile of group 1 mGluRs and their

endogenous regulators, and determine how this profile is altered in the perinatal
PCP rodent model of schizophrenia.

4. To investigate the short- and long-term effects of chronic adolescent CDPPB

treatment on glutamatergic receptor expression within the frontal cortex and
hippocampus.

1.2.3 Rationale and Hypotheses

1. Recent evidence from the McCollum group has indicated glutamaterigic
abnormalities in the nucleus accumbens in schizophrenia, including reports of
altered protein expression of the glutamate vesicle transporter, VGLUT2 and
glutamate projections onto the nucleus accumbens. Furthermore, our previous
findings show altered group 1 mGluR expression in the dorsolateral prefrontal
cortex and hippocampus, regions that project onto the nucleus accumbens.

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 Therefore, it was hypothesised that glutamatergic receptors would be disrupted
in the nucleus accumbens in schizophrenia.

2. The extensive work from de Bartolomeis and colleagues reports typical and
atypical antipsychotics can alter mRNA expression of striatal mGluR5 and its
endogenous regulator, Homer1, in particular Homer1a. Therefore, it was
hypothesised that typical and atypical antipsychotics would alter protein
expression of group 1 mGluRs and Homer1 expression in the nucleus accumbens.

3. Despite group 1 mGluR subtypes sharing many commonalities in structure and

G-protein coupling and pharmacology, recent evidence highlights differences
between the localisation, downstream signalling and receptor interactions
between mGluR1 and mGluR5. These variances are supported by previous
evidence indicating divergent mRNA transcript expression throughout
neurodevelopment and following acute administration of NMDA receptor
antagonists. Therefore, it was hypothesised mGluR1 and mGluR5 protein levels
will exhibit unique patterns of neurodevelopmental expression, which will
perinatal PCP treatment.

4. Acute and chronic administration of the mGluR5 PAM, CDPPB, in adult and
adolescent rodents has shown cognitive enhancing effects. Furthermore, the
behavioural effects of adolescent CDPPB administration have shown to extend
into adulthood, weeks after the cessation of treatment. In adult rodents treated
with CDPPB, there is evidence these behavioural effects are mediated via
CDPPB’s ability to upregulate glutamatergic signalling, in particular via the NMDA
and AMPA receptor. Therefore, it was hypothesised chronic adolescent CDPPB
treatment will have short- and long-term effects on glutamatergic signalling.
Specifically, due to the known interaction of mGluR5 and the NMDA and AMPA
receptor, CDPPB will change the expression of these receptors.

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1.11 Significance
Schizophrenia is a chronic and debilitating disorder that affects approximately 1% of the
world’s population. Current treatments for schizophrenia primarily target the
dopaminergic system, particularly within the nucleus accumbens. However, these
compounds do not adequately treat all symptoms. The absence of an effective
treatment for schizophrenia is largely due to a lack of understanding regarding the
neurochemical underpinnings of the disorder. There is growing evidence that
disturbances to the development and function of the glutamatergic system could
contribute to the pathogenesis of schizophrenia.

This thesis addresses whether the family of glutamatergic receptors, group 1 mGluRs
and their expression in schizophrenia, particularly within the nucleus accumbens is
altered and if they are affected following treatment with antipsychotic medication.
Furthermore, this thesis will investigate the neurodevelopmental expression of group 1
mGluRs within a rodent model of schizophrenia and provide an understanding of how
group 1 mGluRs may participate in the pathogenesis of schizophrenia. In addition, this
neurodevelopmental approach may identify potential neurodevelopmental windows
where targeting these receptors, in particular mGluR5, may be therapeutically
advantageous. Finally, this study will examine the neurochemical effect of adolescent
CDPPB treatment to assist with the molecular understanding of mGluR5-targeting
compounds and aid their preclinical development in an effort to find novel and effective
treatment strategies for schizophrenia.

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 Chapter Two

2.1 Study rationale and aims

Previous immunoblot investigation of postmortem tissue from our laboratory indicate
increased mGluR1 and mGluR5 protein expression in the hippocampus of schizophrenia
subjects. In addition, we also report schizophrenia subjects exhibit increased mGluR5
protein expression in the dorsolateral prefrontal cortex. Furthermore, previous
postmortem investigation of the dorsolateral prefrontal cortex and hippocampus have
also reported NMDA receptor deficits, further supporting altered glutamatergic
signalling to these brain regions. These regions send projections to the nucleus
accumbens, which recent postmortem studies suggests exhibits glutamatergic
alterations associated with schizophrenia. Chapter 2 aimed to explore if the recent
glutamatergic alterations reported in the nucleus accumbens of schizophrenia subjects
extends to the protein expression of group 1 mGluRs and NMDA receptors.

2.2 Manuscript details

The results of Chapter 2 are published in the Journal of Psychiatry and Neuroscience,
entitled “A postmortem analysis of NMDA ionotropic and group 1 metabotropic
glutamate receptors in the nucleus accumbens in schizophrenia”.

2.3 Author contributions

J. Lum performed the experiments, acquired and analysed the data, wrote the first draft
of the manuscript, which all authors reviewed and approved for publication.

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2.4 Collaborator’s statement
We hereby declare that the statement in Section 2.3 pertaining to the contributions of
J.Lum is correct.

Samuel Millard

Xu-Feng Huang

Lezanne Ooi

Kelly Newell

Jeremy Lum 72

Article below removed for copyright reasons, please refer to the citation:

Lum JS, Millard SJ, Huang XF, Ooi L, Newell KA, 2017. A postmortem analysis of
NMDA ionotropic and group 1 metabotropic glutamate receptors in the nucleus
accumbens in schizophrenia. Journal of Psychiatry and Neuroscience. 42, 170077.
DOI: 10.1503/jpn.170077
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 Chapter Three

3.1 Study rationale and aims

The results from Chapter 2 indicate the protein expression of group 1 mGluRs is not
altered in the nucleus accumbens of a large schizophrenia postmortem cohort.
However, previous evidence indicates commonly used antipsychotic compounds can
alter glutamatergic tone in the nucleus accumbens, indicating the results of Chapter 2
may be influenced by premortem antipsychotic use. Further supporting this notion, a
large body of work in rodents, suggest antipsychotics may alter group 1 mGluR signalling
and localisation, as administration of the typical and atypical antipsychotics, haloperidol
and aripiprazole, have shown to alter the mRNA expression of group 1 endogenous
regulators such as Homer1. However, it has yet to be examined if this translates into
changes to respective protein levels. Chapter 3 aimed to investigate the effect of short-
and long-term antipsychotic treatment on the protein expression of group 1 mGluRs and
their endogenous regulators (Homer1a, Homer1b/c and Norbin) in the nucleus
accumbens of a rodent model.

3.2 Manuscript details

The results of Chapter 3 are published in Psychiatry Research, entitled “Effects of short-
and long-term aripiprazole treatment on group 1 mGluRs in the nucleus accumbens:
Comparison with haloperidol”.

3.3 Author contributions

J. Lum performed the experiments, acquired and analysed the data, wrote the first draft
of the manuscript, which all authors reviewed and approved for publication.
Antipsychotic drug treated rat tissue was provided by B. Pan and C. Deng, which J. Lum
analysed.

Jeremy Lum 91
3.4 Collaborator’s statement
We hereby declare that the statement in Section 3.3 pertaining to the contributions of
J.Lum is correct.

Bo Pan

Chao Deng

Xu-Feng Huang

Lezanne Ooi

Kelly Newell

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 Chapter Four

4.1 Study rationale and aims

The results from Chapter 2 and previous postmortem results reported by our laboratory
indicate dysregulation of group 1 mGluRs in schizophrenia may be localised to the
prefrontal cortex and hippocampus (Matosin et al., 2015b, 2015c, 2016a), rather than
the nucleus accumbens. Therefore, the following Chapters focused on exploring the role
of group 1 mGluRs in the prefrontal cortex and hippocampus in the pathogenesis and
treatment of schizophrenia.

Postmortem tissue provides a valuable tool to investigate pathological alterations

associated with schizophrenia, however only provides a snapshot of the end stage of
disease. The pathogenesis of schizophrenia is widely believed to be
neurodevelopmental in origin and derived from glutamatergic alterations through
critical stages of neurodevelopment. Group 1 mGluRs play an integral role in many
neurodevelopmental processes, many of which are thought to be disrupted in
schizophrenia. In an attempt to understand the neurodevelopmental role of group 1
mGluRs and their implication in neurodevelopmental disorders, several studies have
investigated the protein expression of group 1 throughout human and rodent
neurodevelopment using immunoblotting techniques. However, previous studies have
examined group 1 mGluRs under reducing conditions and only examined their
respective monomeric bands and disregarding the dimeric form, which is now widely
believed to be their functional unit. Chapter 4 aimed to investigate the protein
expression of group 1 mGluRs, in particular their dimeric and monomeric form, in the
frontal cortex and hippocampus at critical stages of neurodevelopment (juvenile,
adolescence and adulthood) and how this may be altered in the perinatal PCP model of
schizophrenia.

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4.2 Manuscript details
The results of Chapter 4 are published in Scientific Reports entitled,
“Neurodevelopmental expression profile of dimeric and monomeric group 1 mGluRs:
Relevance to schizophrenia pathogenesis and treatment”.

4.3 Author contributions

J. Lum performed the experiments, acquired and analysed the data, wrote the first draft
of the manuscript, which all authors reviewed and approved for publication. Rodents
were treated and tissues obtained by K. Newell, N. Matosin, F. Fernandez and J.
Andrews; brain tissue was processed and analysed by J. Lum.

4.4 Collaborator’s statement

We hereby declare that the statement in Section 4.3 pertaining to the contributions of
J.Lum is correct.

Francesca Fernandez

Natalie Matosin

Jessica Andrews

Xu-Feng Huang

Lezanne Ooi

Kelly Newell

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 Chapter Five

5.1 Study rationale and aims

Recent evidence indicates mGluR5-stimulation, with PAMs such as CDPPB, may provide
a novel therapeutic strategy for the treatment of schizophrenia and other
neurodevelopmental disorders such as autism. Early intervention is proposed to be
therapeutically advantageous for schizophrenia and other neurodevelopmental
disorders. The results from Chapter 4 indicate monomeric mGluR5 expression is
relatively high during early neurodevelopmental periods and may not be a suitable
therapeutic window for PAM administration, as it may produce agonist-like activity.
However, adolescence may provide a more suitable therapeutic window for PAM
intervention. In line with this, recent studies have shown mGluR5 PAM administration
during adolescence can prevent schizophrenia-like behaviours into adulthood. However,
there is little molecular understanding of the short- and long-term effects of mGluR5
PAMs, which may underlie their therapeutic efficacy. Chapter 5 aimed to explore the
effect of adolescent CDPPB treatment on glutamatergic receptor expression in the
frontal cortex and hippocampus of adolescent and adult rodents.

5.2 Manuscript details

The results of Chapter 5 are published in Neurochemical Research entitled, “Chronic
adolescent CDPPB treatment alters short-term, but not long-term, glutamatergic
receptor expression”.

5.3 Author contributions

J. Lum performed the experiments, acquired and analysed the data, wrote the first draft
of the manuscript, which all authors reviewed and approved for publication.

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5.4 Collaborator’s statement
We hereby declare that the statement in Section 5.3 pertaining to the contributions of
J.Lum are correct.

Samuel Millard

Elisabeth Frank

Natalie Matosin

Xu-Feng Huang

Lezanne Ooi

Kelly Newell

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Article below removed for copyright reasons, please refer to the citation:

Lum JS, Millard SJ, Frank E, Matosin N, Huang XF, Ooi L, Newell KA, “Chronic
adolescent CDPPB treatment alters short-term, but not long-term, glutamatergic
receptor expression”, In Press, Neurochemical Research. DOI: 10.1007/s11064-
018-2584-x
 Chapter Six

6.1 Overall discussion

The past five decades of schizophrenia research have strongly pointed to disrupted
development of the glutamatergic system, in particular the NMDA receptor, in the
pathogenesis of schizophrenia. More recently, evidence from human and animal studies
supports a role for the group 1 mGluRs, which physically and functionally interact with
the NMDA receptor (Matosin and Newell, 2013). In line with this, current efforts to
develop group 1 mGluR-targeting compounds, in particular, mGluR5, are being pursued
as a novel therapeutic for schizophrenia, in addition to other neurodevelopmental
disorders. The present work aimed to investigate the role of group 1 mGluRs in the
development and pathophysiology of schizophrenia, as well as examining the
neurochemical changes induced by the mGluR5 PAM, CDPPB.

Postmortem tissue provides an important tool to investigate and understand the

pathology of neurological and neuropsychiatric disorders. Evidence from postmortem
studies investigating the protein expression of group 1 mGluR has been varied and
limited to elderly cohorts and low-powered sample size (summarised in Section 1, Table
1.2). However, our research group recently reported increased protein expression of
mGluR5 in the dorsolateral prefrontal cortex and altered mGluR1 and mGluR5 levels in
the hippocampus of adequately powered postmortem cohorts (Matosin et al., 2015b,
2015c, 2016a). Although the aforementioned studies were performed under reducing
conditions, which can reduce dimeric mGluR disulphide bonds to their respective
monomeric forms, these changes were greater or specifically related to the dimeric form
of mGluR1 or mGluR5. Interestingly, recent evidence indicates the dimeric form of
mGluRs to be the functional signalling unit (El Moustaine et al., 2012).

In an effort to follow on from our previous studies and address dimeric/monomeric

protein levels, Chapter 2 sought to investigate the protein expression of group 1 mGluR
monomeric and dimeric forms in the nucleus accumbens of a schizophrenia postmortem

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cohort. As discussed in Chapters 1 and 2, whilst the nucleus accumbens has historically
been associated with dopaminergic abnormalities in schizophrenia, recent in vivo
imaging studies indicate these abnormalities may not be as prominent as previously
thought (Howes et al., 2009; Kegeles et al., 2010). Furthermore, recent postmortem
evidence suggests the nucleus accumbens exhibits altered glutamatergic tone
(McCollum et al., 2016; McCollum and Roberts, 2015). The nucleus accumbens is a
region of convergence for the dorsolateral prefrontal cortex and hippocampus, where
we previously reported altered group 1 mGluR expression. Furthermore, prefrontal
cortical glutamatergic afferents onto the nucleus accumbens signal via mGluR1 and
mediate motivational behaviour (Turner et al., 2018), a common symptom reported in
schizophrenia patients. Utilising the largest nucleus accumbens postmortem cohort to
date, the present study reported no significant change in the total, dimeric or
monomeric protein expression of either mGluR1 and mGluR5 in schizophrenia subjects,
in line with a previous study investigating total mGluR1 and mGluR5, in a smaller cohort.
In addition, no differences were observed in the protein expression of the group 1
mGluR endogenous regulators, Norbin and Homer1b/c. Furthermore, there was also no
difference in the protein expression of NMDA receptor subunits, NR1, N2A and NR2B, in
line with previous mRNA and binding studies (Aparicio-Legarza et al., 1998; Meador-
Woodruff et al., 2001; Noga et al., 1997). Collectively, the present and previous results
examining group 1 mGluRs, NMDA receptors and other glutamatergic receptors in the
nucleus accumbens, do not support the recent findings from McCollum and colleagues
suggesting glutamatergic abnormalities in this region. However, it should be noted that
McCollum reported an increase of glutamatergic synapses specifically within the core
and not shell of the nucleus accumbens, whereas the present study and previous studies
have not separated these subregions. Whilst neither study strongly supports nor rejects
glutamatergic dysfunction within the nucleus accumbens, further studies should be
undertaken to explore this notion.

A large majority of schizophrenia postmortem studies report positive findings in the

dorsolateral prefrontal cortex and hippocampus. However, this may be largely due to
the fact these are areas most extensively investigated. Furthermore, a limititation of

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many postmortem studies is the absence of follow-up investigations in other brain
regions to determine if observed alterations are global or selective. We previously
reported increased mGluR5 expression in the dorsolateral prefrontal cortex and
hippocampus of schizophrenia subjects (Matosin et al., 2015b, 2015c). Chapter 2
followed on from these findings to examine mGluR5 expression in the nucleus
accumbens, a brain region that receives dense innervation from both the dorsolateral
prefrontal cortex and the hippocampus (Britt et al., 2012). A significant number of
subjects analysed in our previous studies were included in Chapter 2 (Table 6.1),
providing the opportunity to examine if mGluR5 expression was correlated between
brain regions and identify if alterations in projecting regions, may influence nucleus
accumbens expression. It was postulated that mGluR5 expression might be increased in
the nucleus accumbens of subjects that exhibited increased expression in the
dorsolateral prefrontal cortex and hippocampus, indicating a global increase in mGluR5
levels. However, Spearman’s correlations revealed no significant association of mGluR5
protein levels between the brain regions (Table 6.2). Furthermore, analysis of mGluR5
expression in the dorsolateral prefrontal cortex, hippocampus and nucleus accumbens
of the same subjects revealed no significant diagnostic difference in the nucleus
accumbens (Figure 6.1a), despite this subset of schizophrenia subjects still exhibiting
significantly increased mGluR5 expression in the dorsolateral prefrontal cortex and
hippocampus (Figure 6.1b-c), as previously reported (Matosin et al., 2015b, 2015c). This
indicates that mGluR5 expression is not globally increased in schizophrenia and in line
with many other studies that suggest glutamatergic changes are largely present in
cortical and hippocampal regions with scarce reports of changes in the nucleus
accumbens. It is unclear, why the dorsolateral prefrontal cortex and hippocampal
regions may be more sensitive to glutamatergic alterations or why the nucleus
accumbens is immune, particularly considering many cortical and hippocampal
glutamatergic efferents project onto the nucleus accumbens. However, the reason may
lie in the cytoarchitectural heterogeneity between the regions. The dorsolateral
prefrontal cortex and hippocampus are highly comprised of pyramidal cells and
interneurons (Lewis, 2004; Slomianka et al., 2011), whilst the nucleus accumbens
primarily consists of medium spiny neurons (Britt et al., 2012). Our studies to date have

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yet to investigate if a specific cell type(s) may be responsible for the observed increases
in mGluR5 expression within the dorsolateral prefrontal cortex and hippocampus,
however obtaining this knowledge would aid in further dissecting the underlying
pathology of schizophrenia. Nevertheless, it seems dysregulation of mGluR5 may not
apply universally or influence the expression of mGluR5 in projecting regions, however
it should be considered whether it is localised to specific cell types; further examination
on this, preferably within the same cohort of subjects, is warranted.

Table 6.1: Summary of postmortem subject demographics included from our

laboratory’s analysis of the nucleus accumbens, dorsolateral prefrontal cortex and
hippocampus (cornu ammonis 1).
Control Schizophrenia
Variable t p
(n=16) (n=13)
Age at death (years) 58.75±13.72 52.46±13.93 1.219 0.233
Postmortem interval (hours) 25.75±12.78 29.65±11.44 -0.857 0.399
Brain pH 6.53±0.25 6.56±0.24 -0.338 0.738
Freezer Storage time (days) 3753±871 4147±617 -1.421 0.167
Hemisphere 11 Right, 5 Left 8 Right, 5 Left
Gender 14M, 2F 7M, 6F
Values are represented as mean±SEM. M: males; F: Females.

Table 6.2: Spearmans’s correlations for associations of total mGluR5 protein levels between
the nucleus accumbens (NAcc), dorsolateral prefrontal cortex (DLPFC) and hippocampus
(cornu ammonis; CA1)
Brain Region NAcc DLPFC CA1

NAcc r= -0.313 r= -0.104

---------------
p= 0.105 p= 0.592

DLPFC r= -0.313 r= 0.321

---------------
p= 0.105 p= 0.096

CA1 r= -0.104 r= 0.321

---------------
p= 0.592 p= 0.096

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Figure 6.1: Total metabotropic glutamate receptor 5 (mGluR5) protein levels in the a)
nucleus accumbens, b) dorsolateral prefrontal cortex and c) hippocampus (CA1; cornu
ammonis 1) between schizophrenia (squares) and control groups (circles). *p<0.05 and
*** p<0.001.

Despite the lack of glutamatergic changes observed in Chapter 2, the nucleus accumbens
is still highly regarded as a region of interest within schizophrenia research, due to
evidence indicating it is a target area for mediating the actions of dopaminergic
antipsychotic drugs. Whilst the results of Chapter 2 indicated no evidence of altered
group 1 mGluR protein expression in schizophrenia, we cannot disregard the potential
effect of premortem antipsychotic medication within the schizophrenia cohort. The
daily lifetime chlorpromazine equivalent value for each subject is often used to associate
the relationship between premortem antipsychotic use and investigated variables. In
this study, daily lifetime chlorpromazine equivalent values showed no association with
protein levels of group 1 mGluRs or NMDA receptor subunits, neurochonrin or
Homer1b/c protein levels. Chlorpromazine equivalent value is defined as the dosage of
antipsychotic, which has equivalent D2 receptor occupancy and antipsychotic potency
to 100 mg of chlorpromazine (Woods, 2003). Whilst chlorpromazine equivalent values
can provide an indication of premortem antipsychotic use, these results should be
interpreted with caution, for several reasons. Equivalent values are largely based on D2
receptor occupancy and do not take into account the occupancy of other receptor types,
which for atypical antipsychotics, are thought vital for their efficacy. Furthermore, the
correlation between antipsychotic dose and antipsychotic efficacy may not be linear, as
evidence suggests the relative potency of the typical antipsychotic, haloperidol declines
as the dose increases (Foster, 1989). For this reason, animal models can provide a useful
tool to not only dissect the mechanisms of actions of these compounds, but also to aid

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our understanding of whether postmortem human findings are a true indication of
disease pathology or may be the result of premortem antipsychotic treatment.

The results of Chapter 3 showed that short- and long-term treatment with the typical
and atypical antipsychotics, haloperidol and aripiprazole, did not alter group 1 mGluR
protein levels in the nucleus accumbens in a rodent model. Previous examination of this
model has shown NR1 protein expression to be upregulated in the nucleus accumbens
following haloperidol and aripiprazole treatment (Pan et al., 2016). This is in line with
similar results suggesting glutamatergic tone in the nucleus accumbens is altered
following antipsychotic treatment (Fitzgerald et al., 1995; Healy and Meador-Woodruff,
1997; Pan et al., 2016; Schmitt et al., 2003; Spurney et al., 1999). Particularly, previous
evidence indicates ionotropic glutamate receptors are more prone to expression
changes following antipsychotic treatment, whilst there is little evidence to suggest
changes to group 1 mGluRs. This is unexpected, considering the evidence supporting a
physical and functional link between D2 receptors and group 1 mGluRs, in particular,
mGluR5 (Mao and Wang, 2016). Furthermore, agonist stimulation of mGluR5 has been
shown to reduce D2 receptor agonist binding, suggesting a close functional association
(Servaes et al., 2017). Despite, the lack of change in protein expression of group 1
mGluRs reported in Chapter 3, Homer1a and Norbin expression were increased
following antipsychotic treatment. The present study is the first to examine the protein
expression of Homer1 following antipsychotic treatment. However, the present results
are in line with studies from de Bartolomeis and colleagues that have repeatedly
reported changes to Homer1 mRNA expression following antipsychotic treatment
(Ambesi-Impiombato et al., 2007; de Bartolomeis et al., 2002, 2013; Iasevoli et al.,
2010a, 2010b, 2011; Tomasetti et al., 2007). Homer1 and Norbin have been shown to
regulate the localisation and signalling of group 1 mGluRs. This may indicate that whilst
group 1 mGluR protein levels are unchanged, their localisation and signalling in the
nucleus accumbens may be altered following antipsychotic treatment. However, further
studies investigating the interaction of these proteins and the potential biological
consequences will be required to understand the potential effect of antipsychotic
treatment for group 1 mGluR signalling and if this mediates the action of antipsychotics.

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Collectively, Chapters 2 and 3 aimed to further build on previous findings from our
laboratory regarding altered expression of group 1 mGluRs and investigate if similar
alterations were observed in the nucleus accumbens, a region highly implicated in the
pathogenesis of schizophrenia. However, the findings presented here provide little
support of similar alterations that we had previously observed in the dorsolateral
prefrontal cortex and hippocampus, indicating brain-region specific differences. Whilst
the present data cannot eliminate the possibility that glutamatergic alterations exist in
the nucleus accumbens, previous evidence from our own lab and others provide
stronger support for glutamatergic alterations in cortical and hippocampal regions. In
line with this, the majority of previous examinations of group 1 mGluRs in schizophrenia
have focused on its implication in associated cognitive deficits. Furthermore,
investigation of mGluR5 as a novel therapeutic target has largely focused on its ability
to enhance cognitive processes, primarily mediated by cortical and hippocampal
regions, collectively suggesting group 1 mGluRs may play a larger role in these regions,
rather than subcortical regions, like the nucleus accumbens. Based on these brain-region
specific effects, Chapters 4 and 5 focused on the role of cortical and hippocampal group
1 mGluRs in regards to the pathology and treatment of schizophrenia.

Chapter 4 explored the neurodevelopmental expression of group 1 mGluRs, specifically

in the perinatal PCP model, a well-established neurodevelopmental model of
schizophrenia. This model has previously shown to produce alterations in NMDA
receptor expression and phosphorylation, a reflection of altered glutamatergic signalling
and in line with the glutamatergic hypothesis of schizophrenia (Anastasio and Johnson,
2008a, 2008b; du Bois et al., 2012; Wang et al., 2001). Analysis of group 1 mGluRs in this
model revealed mGluR1 dimer expression was reduced in the hippocampus at PN12,
following perinatal PCP exposure, whilst mGluR5 protein levels were increased at the
same time-point in the frontal cortex and hippocampus. As previously, described
mGluR5 has a strong physical and functional link with the NMDA receptor and was
previously shown to upregulate NMDA receptor activity. Furthermore, early postnatal
administration with the NMDA receptor antagonist, MK-801 has previously shown to

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acutely increase mGluR5 mRNA expression (Wilson et al., 1998). Collectively, this may
suggest mGluR5 expression/activity may be upregulated following NMDA receptor
blockade, as a compensatory mechanism. Interestingly, the upregulation of mGluR5
protein was only observed to the dimeric form, which is known to be the functional
signalling unit for mGluRs, highlighting the importance of measuring this form.

A growing body of evidence clearly supports that mGluR dimerisation is vital for
traditional agonist-induced downstream signalling (El Moustaine et al., 2012; Kniazeff et
al., 2004). Therefore, throughout this thesis, particular effort was made to quantify
mGluR1 and mGluR5 protein expression under non-reducing conditions, to ensure
quantification of the dimeric bands. Immunoblots of mGluR1 or mGluR5 under non-
reducing conditions exhibited three separate bands. One band was observed at
approximately 150kDa, representing the monomeric form of the receptors.
Furthermore, two distinct bands were observed and migrated at approximately 270-280
kDa represent the dimeric form of these receptors. To validate the specificity of these
dimeric bands, two additional antibodies for mGluR1α and mGluR5, (MAB07-617 and
Ab5675, respectively) were used. These antibodies recognise separate epitopes than the
antibodies used throughout this thesis however, showed a similar pattern of binding
where two dimeric bands were observed for mGluR1 and mGluR5 (Figure 6.2a-b). This
provides strong evidence that these bands do represent specific mGluR1 and mGluR5
proteins. Furthermore, the presence of these two distinct dimeric bands has previously
been reported by others and specificity confirmed in respective KO mice (Ayala et al.,
2012; Kirschstein et al., 2007; Lee et al., 2015). However, it is unclear what these two
dimeric bands may represent. These two dimer bands are unlikely to represent splice
variants, as although mGluR5 has two isoforms of similar molecular weight (mGluR5a;
128 kDa and mGluR5b; 132 kDa), mGluR1 does not have a splice variant of a similar
molecular weight to mGluR1α (132 kDa) (Ferraguti et al., 2008). However, post-
translational modifications including ubiquitination, phosphorylation, palmitylation and
glycosylation can cause proteins to migrate differently to their predicted molecular
weight (Bass et al., 2017). These post-translational modifications can influence a
protein’s function, trafficking and localisation and therefore are of value to investigate.

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In fact, the glycosylation of several glutamatergic proteins including the AMPA receptor
subunit, GluA2 and excitatory amino acid transporters, EAAT1 and EAAT2 have been
reported to be altered in schizophrenia (Bauer et al., 2010; Tucholski et al., 2013).
mGluR1/5 contain several N-linked glycosylation sites, particularly at the N-terminus,
that could increase their expected molecular weight and may subsequently represent
the two dimer bands (glycosylated and glycosylated mGluR1/5) (Bhave et al., 2003).
However, incubation of tissue with the N-linked deglycosylation enzyme, Peptide:N-
Glycosidase F (PNGase F) and subsequent immunoblot of mGluR5, showed both dimeric
bands were sensitive to PNGase treatment (Figure 6.2c). Whilst this does not suggest
the bands represent different N-linked glycosylation status, it does not eliminate other
forms of glycosylation or post-translational modifications. Further investigation to
determine the exact nature of these bands may highlight differences in the function,
trafficking or localisation of group 1 mGluRs.

Figure 6.2: Immunoblots of rodent cortical tissue with a. mGluR1 (MAB07-617) and b.
mGluR5 (ab5675), show two bands (indicated by arrows) at a molecular weight
corresponding to the molecular weight of the dimeric mGluR1α/5, similar to the pattern
observed from the antibodies used throughtout this thesis. c. Adult rat cortical
homogenate was incubated with the deglycosylation enzyme, PNGase F (1 or 10
units/10 ug homogenate) for 3 hours. PNGase F treated homogenate was then
separated by electrophoresis and proteins were transferred onto PDVF membrane.
Membranes were subsequently incubated with anti-mGluR5 antibody (ab27190).
Immunoblots revealed that pre-treatment of rat tissue PNGase F (10 units), caused a
molecular weight shift in both dimeric bands of mGluR5. kDa, kilodaltons.

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Whilst there have been several studies investigating the neurodevelopmental profile of
group 1 mGluRs, none to date were performed under non-reducing conditions and
investigated the dimeric:monomeric form of these proteins. Chapter 4 clearly shows the
importance of examining this, as it was evident that the monomeric form of mGluR5 is
highly abundant at PN12 in all brain regions examined (hippocampus, frontal cortex and
nucleus accumbens), compared to the later time points examined (PN35 and PN96),
where it was almost undetectable, irrespective of perinatal PCP treatment. This is the
first report of an mGluR, showing such a stark neurodevelopmental change in
dimeric:monomeric expression. Furthermore, it appeared to be restricted to mGluR5, as
analysis of mGluR1 did not show a similar trend. However, it is also unclear why we
observed this trend, as dimerisation of mGluRs is complex and still not well understood.
However, it is established that mGluR5 dimerises in the endoplasmic reticulum and Golgi
apparatus (Pin et al., 2003). The high levels of monomeric mGluR5 observed during the
juvenile period may represent newly synthesised and immature mGluR5 within the
ribosomes, endoplasmic reticulum or Golgi apparatus, in preparation for post-
translational modification or heterodimerisation with other G-protein coupled
receptors such as mGluR1, D2, A2A and calcium-sensing receptors. Furthermore, it may
represent functional mGluR5 within a specific subcellular compartment, as work from
Karen O’Malley’s group have clearly demonstrated mGluR5 is expressed on organelle
membranes and plays a signalling role, including within the nuclear membrane (Jong et
al., 2009; Jong et al., 2014; Kumar et al., 2008; O’Malley et al., 2003b; Sergin et al., 2017).
Whilst the current study did not examine the expression of mGluR5 within subcellular
fractions, it does not appear that mGluR5 monomer is highly abundant within the
nuclear membrane (personal communication with Karen O’Malley); however, this is yet
to be explored on other organelle membranes. Furthermore, recent evidence from our
laboratory also showed that this high mGluR5 monomer expression is not specific to
Sprague-Dawley rats, as we observed a similar trend in the Wistar-Kyoto strain of rats
(Figure 6.3). However, it is unknown if this pattern of expression is conserved in other
species. It would be of particular interest to examine this in a neurodevelopmental
human postmortem cohort, particularly as it may inform the potential use of mGluR5
pharmaco-therapeutics during neurodevelopmental periods. As previously discussed,

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dimerisation of mGluRs is vital for agonist-induced signalling, furthermore dimerisation
appears integral for controlling PAM downstream activity. Whilst monomeric mGluRs
are still able to couple to G-proteins, they are unable to elicit a downstream effect in the
presence of the agonist, glutamate. However, the presence of PAMs was able to
stimulate downstream activation of monomeric mGluR, in the absence of glutamate (El
Moustaine et al., 2012). This could pose a major limitation to mGluR5 PAM
development, as a major advantage of PAMs is their ability to only potentiate a response
in the presence of an orthosteric ligand and avoid constant and elongated receptor
stimulation, which is known to cause receptor desensitisation, excitotoxcity and
seizures. Consequently, if PAMs were administered during a neurodevelopmental
period of high monomeric mGluR5 expression, this could cause a wide array of
neurotoxic effects. In fact, mGluR5 PAMs have been proposed to treat a wide range of
neurodevelopmental disorders, such as autism, Rett syndrome and schizophrenia,
where early intervention is thought to be advantageous. However, based from the
evidence presented in Chapter 4, it is proposed mGluR5 PAM administration during early
neurodevelopmental periods may have potential harmful effects. Therefore, further
investigation of mGluR5 PAM administration during the juvenile period is warranted to
examine for potential induction of cell death and excitotoxicity processes.

Figure 6.3: Representative immunoblot images of mGluR5 in the frontal cortex of

Sprague-Dawley (SD) and Wistar-Kyoto (WKY) rats at postnatal days (PN) 12, 35 and 96.
kDa, kilodaltons.

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The basis of mGluR5’s therapeutic potential for schizophrenia was originally thought to
be due to its ability to modulate NMDA receptor activity (Kinney et al., 2005). In
particular, administration of mGluR5 PAMs at adulthood, have shown to attenuate
various schizophrenia-like behaviours in animal models. Based on the evidence in
Chapter 4, we hypothesised targeting mGluR5 at adolescence may provide a beneficial
treatment window, as there is little expression of monomeric mGluR5 at this timepoint
and this age period when schizophrenia symptoms commonly begin to emerge.

Chapter 5 aimed to investigate the short- and long-term effects of chronic adolescent
CDPPB treatment, on glutamatergic receptor expression in the prefrontal cortex and
hippocampus, two regions implicated in schizophrenia and involved in cognitive
processes. Our study showed a general upregulation of the glutamatergic receptor
proteins measured in the adolescent hippocampus, which included mGluR5, the NMDA
receptor subunits, NR1, NR2A and NR2B and the AMPA receptor subunits GluA1 and
GluA2. However, no changes to these proteins were observed in the adolescent
prefrontal cortex, suggesting brain region specific effects of chronic adolescent CDPPB
treatment. In line with this, previous studies have also reported CDPPB treatment
caused brain region specific effects following acute and/or chronic administration at
adulthood (Parmentier-Batteur et al., 2012; Uslaner et al., 2009). Whilst it is unclear
why CDPPB may have brain region specific effects, mGluR5 displays diverse cell type
expression, protein interactions and subsequent signalling in different brain regions
(Matosin et al., 2016), which may underlie the brain specific changes observed in
Chapter 5. Further understanding the unique patterns of mGluR5 signalling and
neurochemical changes caused specifically by various mGluR5 PAMs within certain brain
regions, may inform drug discovery efforts for the variety CNS disorders mGluR5 PAM
treatment has been proposed.

Despite observing changes in the adolescent hippocampus following CDPPB treatment,

similar changes at adulthood in the hippocampus or prefrontal cortex were not
observed, indicating the current treatment model did not cause long-term changes to

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the glutamatergic signalling, at least under the current treatment paradigm. In line with
this, Parmentier-Batteur (2012) reported adult rats treated with daily CDPPB treatment
at the same dose used in the present study for seven days was able to attenuate
amphetamine-induced hyperlocomotive deficits. However, a group of rats treated for
six days and subsequently withdrawn from treatment for one day did not show the same
treatment effect, suggesting the effects of chronic CDPPB treatment are reversible.
Using a lower dose of CDPPB (10 mg/kg), a previous study investigating the effect of
chronic adolescent CDPPB treatment reported an ability to attenuate neonatal PCP
induced social interaction behavioural deficits several weeks following the cessation of
treatment (Clifton et al., 2013). Acute dosage studies indicate CDPPB displays an
inverted U-shaped dose effect on cognitive performance and NMDA/AMPA receptor
phosphorylation, consequently leading to the thought a lower dose of CDPPB such as 10
mg/kg, might be more beneficial, than a higher 30 mg/kg dose (Uslaner et al., 2009).
Whilst no study to date has examined a dose-dependent study for chronic
administration of mGluR5 PAMs, it could be postulated that chronic treatment with a
lower dose of 10 mg/kg will produce greater effects on behaviour and glutamatergic
signalling, than the 30 mg/kg dose used in the present study. The dose chosen in this
thesis was based on previous studies indicating this dose enhanced performance in
spatial learning tasks and ameliorated NMDA receptor antagonist-induced cognitive set-
shifting deficits (Fowler, 2012; Stefani and Moghaddam, 2010). However, it should be
noted these studies used an acute dose of CDPPB, with no study to date investigating a
dose response in a chronic treatment paradigm. Whilst results from the present study
do not support that adolescent CDPPB treatment causes long-term changes in regards
to glutamatergic receptor expression, this does not exclude that short- or long-term
changes could have occurred to other neurotransmitter or associated systems. CDPPB
was shown to affect a wide array of neurotransmitter systems, including the
dopaminergic and GABAergic systems, which are of high relevance to schizophrenia. A
further understanding of the molecular and dose effects of CDPPB and other mGluR5
PAMs will help improve drug design and development.

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A large majority of studies investigating mGluR5 PAMs, have primarily focused on their
ability to alter neurotransmitter deficits associated with schizophrenia. However, recent
evidence indicates non-neuronal cells may play a more important role in the pathology
of schizophrenia, than previously thought. Postmortem and imaging studies indicate
that a subset of schizophrenia patients exhibit increased inflammatory markers and
reduced hippocampal neural stem cells (Fillman et al., 2013; Reif et al., 2006).
Furthermore, similar findings have also been reported in various animal models of
schizophrenia (Kvajo et al., 2008; Nawa and Takei, 2006), highlighting the importance of
non-neuronal types in the pathology of schizophrenia. mGluR5 is expressed on many cell
types including neural stem cells, oligodendrocytes, astrocytes and microglia (Balázs et
al., 1997; Melchiorri et al., 2007; van den Pol et al., 1995). Activation of mGluR5
expressed on the aforementioned cell types has shown to be responsible for various
functional roles and therefore may be a potential avenue for the therapeutic efficacy of
mGluR5 PAMs, not only for schizophrenia, but also other neurological disorders where
non-neuronal cells may be implicated. The mGluR5 PAM, VU0360172 has shown in vitro
and in vivo evidence to suppress microglial proliferation and activation in a model of
traumatic brain injury (Loane et al., 2014). In addition, evidence also indicates mGluR5
activation is important for the activation of neural stem cell survival and proliferation
within the hippocampus (Jansson and Åkerman, 2014). In line with this, administration
of CDPPB (10 mg/kg daily; i.p) for three days increased the proliferation of neural stem
cells and new neurons within the hippocampus (Nochi et al., 2012). Although,
investigation of the effects of mGluR5 PAMs on glia and neural stem cells is still is in its
infancy, these early studies open an extensive avenue for investigation and may provide
alterative mechanisms for the therapeutic efficacy of mGluR5 PAMs. To date no studies
have directly examined the ability of an mGluR5 PAM to attenuate microglial activation
or neural stem cell proliferation in a rodent model of schizophrenia. However,
considering the evidence supporting a beneficial role of mGluR5 PAMs in these
processes, it warrants investigation and may provide further support for the use of
mGluR5 PAMs in the treatment of schizophrenia.

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6.2 Limitations
Collectively, the work presented in this thesis provides further knowledge of group 1
mGluRs and their endogenous regulators in the pathology and treatment of
schizophrenia. However, several limitations need to be considered.

The present work examined the protein expression of group 1 mGluRs, Norbin and
Homer1 in various paradigms of the pathology and treatment of schizophrenia, by
means of immunoblotting the aforementioned proteins within crude homogenates.
There is now substantial evidence group 1 mGluRs are specifically localised on specific
cell types (Byrnes et al., 2009; Luyt et al., 2003; Romano et al., 1996). Furthermore,
depending on the cell type and proximity to other interacting proteins, such as the
NMDA receptor, group 1 mGluRs signalling can be cell-type dependent. Whilst double
labelling immunohistochemistry may shed light on any potential cell-specific changes of
group 1 mGluR expression, it would not have allowed the examination of
dimer:monomer levels. Recent advances in fluorescence-activated cell sorting or laser-
capture microdissection would allow the separation of cell types and analysis of
dimer:monomer expression, however this approach requires large amounts of tissue,
which is often not possible in human postmortem analyses, and the techniques have
only been successful in a handful of laboratories. In addition to potential cell-type
specific alterations, group 1 mGluRs play a varied role dependent on their subcellular
localisation (Jong et al., 2017). Group 1 mGluRs are localised on various organelle
membranes, most notably the nucleus (Jong et al., 2007, 2005; Sergin et al., 2017).
Whilst this study found no changes in group 1 mGluR protein expression in the nucleus
accumbens of schizophrenia subjects and following antipsychotic treatment, the
present work cannot exclude that there may be changes in their subcellular localisation.
In particular, Chapter 3 shows Homer1a and Norbin, which can regulate mGluR5 cell
surface localisation and downstream signalling, are altered following antipsychotic
treatment. Considering Homer1a and Norbin are responsible for the localisation and
signaling of group 1 mGluRs, particularly mGluR5, it is hypothesised the consequential
upregulation of these endogenous regulators following antipsychotic treatment would
increase group 1 mGluR cell surface expression and downstream signalling.

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Furthermore, antipsychotic treatment has shown to cause synaptic remodelling, in
which group 1 mGluRs and their endogenous regulators play an important role.
Therefore, the upregulation of these proteins may be vital for antipsychotic-induced
synaptic plasticity in the nucleus accumbens. However, future studies are required to
characterise these findings in regards to protein localisation, interaction and potential
biological consequence.

The present work examined both the dimeric and monomeric forms of group 1 mGluRs;
recent evidence indicates group 1 mGluRs can form heterodimers with each other
(Doumazane et al., 2011; Pandya et al., 2016). Furthermore, there is also evidence that
group 1 mGluRs can heterodimerise or at least co-localise and form higher order
oligomer complexes with other GPCRs such as GABAB, D2, calcium-sensing and A2A
receptors, which have also shown to have functional consequences on group 1 mGluR
stimulation. In particular, mGluR5 shares an intriguing complex with A 2A and D2
receptors on striatopalldal GABAergic neurons found projecting from the nucleus
accumbens. Stimulation of mGluR5 has shown to reduce D2 receptor agonist binding
(Popoli et al., 2001). Furthermore, co-stimulation of A2A and mGluR5 decreases agonist
binding to the D2 receptor greater than individual stimulation of either receptor (Popoli
et al., 2001). In addition, perfusion of CHPG and the A2A receptor agonist, CGS 21680,
has shown to increase localised GABA levels far larger than either agonist alone and
similar to level of the D2 receptor antagonist, raclopride (Diaz-Cabiale et al., 2002).
Collectively, it appears mGluR5/A2A/D2 receptors are capable of forming a functional
complex. Whilst the present work did not aim to investigate these potential
heterodimers, examining such heterodimers may provide more insight into the
molecular complexities of psychiatric disorders.

Chapter 5 aimed to investigate the neurochemical effects of adolescent CDPPB

treatment. Whilst, this study provides valuable insight into the short-and long-term
effects on glutamatergic receptor expression, there are several limitations. Firstly, no
behavioural tests were performed to examine if CDPPB treatment showed any cognitive
enhancing effects at adolescence or adulthood, analogous to that reported in previous

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studies (summarised in Table 1.3). The addition of behavioural testing would have
allowed a comparison with the molecular data presented in Chapter 5. Secondly, this
study examined the protein expression of mGluR5, NMDA and AMPA receptor subunits,
which may not accurately affect the active state. Previous studies suggest the
phosphorylation of the receptor may be altered, without changes to protein expression
(Parmentier-Batteur et al., 2012; Uslaner et al., 2009). Furthermore, CDPPB was
administered to naïve animals and not a model of schizophrenia. However, the
development of novel therapeutics requires knowledge of its effects under physiological
and pathophysiological conditions. It is evident that utilising an animal model of
schizophrenia, in addition to a model of autism, Rett syndrome or addiction could cause
neurological changes that may influence the effect of CDPPB treatment on mGluR5
signalling and general glutamatergic neurotransmission. Clifton et al., (2013) reported
promising cognitive effects following adolescent CDPPB treatment within the perinatal
PCP model of schizophrenia, alike the model presented in Chapter 4 of this thesis. The
perinatal PCP model is an established and widely used rodent model of schizophrenia to
examine the underlying neurochemical changes associated with schizophrenia and
investigate the efficacy of novel therapeutic compounds. However, it is unlikely a single
rodent model will encapsulate the heterogeneity of schizophrenia. In line with this,
other models are emerging, which may be beneficial to model the diverse aspects and
origins associated with schizophrenia. Therefore, these parameters should be
considered when examining the efficacy of novel antipsychotics and interpreting
pathological changes in animal models of schizophrenia.

6.3 Future Directions

The present work builds upon a foundation of investigation of group 1 mGluRs and their
involvement in schizophrenia. However, as the field’s understanding of group 1 mGluRs
continues to grow, there are still a multitude of avenues which should be considered for
future studies. It is evident group 1 mGluRs are more complex than simple transmitter
receptors capable of converting extracellular indicators toward intracellular signals.
Recent evidence indicates mGluR5 is found within the nuclear membrane and physically
interacts with chromatin (Sergin et al., 2017). However, the regions of DNA that mGluR5

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may interact with are yet to be investigated, although techniques such as chromatin
immunoprecipitation (CHIP)-seq, could help identify such regions. Furthermore, the
functional consequence of this interaction has not been examined, however previous
proteins identified to interact with chromatin, can regulate transcription and epigenetic
changes to the genome. Epigenetic regulation is a rapidly expanding area of research
and has previously been implicated in schizophrenia and other psychiatric disorders
(Schuebel et al., 2016). Therefore, examination of the mGluR5 and chromatin interaction
would not only provide a further understanding of mGluR5, but also its potential
implication in neurodevelopmental and psychiatric disorders.

Group 1 mGluRs have been implicated in schizophrenia, primarily based on their

interaction with the NMDA receptor (Awad et al., 2000; Doherty et al., 1997, 2000; Jia
et al., 1998; Mannaioni et al., 2001; Pisani et al., 2001). However, group 1 mGluRs have
also shown to interact with the AMPA receptor and regulate their cell surface expression
and activation (Kim et al., 2015; Nakamoto et al., 2007; Ugolini et al., 1999). Previous
studies suggest that NMDA and AMPA receptor trafficking to the cell surface may be
disrupted in schizophrenia, particularly in the cortex (Beneyto and Meador-Woodruff,
2008; Hammond et al., 2010; Kristiansen et al., 2010). Furthermore, based on our
previous investigation of mGluR5 and its endogenous regulators in the dorsolateral
prefrontal cortex, it could be speculated similar trafficking deficits could occur in regards
to mGluR5 (Matosin et al., 2015b). Whilst the present study investigated the expression
of group 1 mGluRs, in conjunction with NMDA and/or AMPA receptor subunit
expression, it did not examine the cellular localisation or direct interaction of these
proteins at the cell surface. To date, no study has examined the cellular localisation of
group 1 mGluRs in schizophrenia postmortem tissue or co-localisation/interaction with
NMDA and AMPA receptors. Future studies investigating such parameters may provide
deeper insight into the role group 1 mGluRs may play in the pathology of schizophrenia.

Norbin has shown to regulate cell-surface expression of mGluR5 (Wang et al., 2009).
Recent evidence indicates mGluR5 localisation to the nuclear membrane is dependent
on the C-terminus region (amino acids 852-876) (Sergin et al., 2017), which is also a

Jeremy Lum 140

region where Norbin interacts with mGluR5, consequently leading to the proposition
Norbin may be involved in the localisation of mGluR5 to the nucleus. Nevertheless, in
vivo and in vitro studies indicate Norbin is important for synaptic plasticity, of which
deficits are observed in many neuropsychiatric disorders (Ohoka et al., 2001; Wang et
al., 2015, 2009). In line with this, aside from group 1 mGluRs, Norbin has shown to
interact with the guanine-nucleotide exchange factor, P-Rex1, which is involved in
regulating cytoskeletal structure, further supporting Norbin’s role in cellular
architecture (Pan et al., 2016). Norbin expression has repeatedly been reported to be
altered in schizophrenia and major depression postmortem cohorts (Martins-de-Souza
et al., 2012; Matosin et al., 2015a, 2015c; Mudge et al., 2008), with the present study
(Chapter 2) being the only study thus far, not to report a pathological change, possibly
related to the subcortical region examined. In addition, Norbin KO mice show
schizophrenia and depression-like behaviours, further supporting its role in
neuropsychiatric disorders (Wang et al., 2015, 2009). Whilst there is little understanding
of its biological function, several questions remain regarding its involvement in
neuropsychiatric disorders. Firstly, does Norbin regulate mGluR5 localisation to the
nuclear membrane and does it regulate the expression of synaptic proteins via such a
manner, as activation of nuclear mGluR5 has shown to activate genes associated with
synaptic plasticity? Secondly, is Norbin’s interaction with mGluR5 critical to its
involvement in synaptic plasticity or does Norbin interact with other synaptic proteins,
which may mediate its effects on neurite outgrowth and synaptic plasticity?
Immunoprecipitation of Norbin and subsequent mass-spectrometry of interacting
proteins would be able to provide some insight. Whilst Norbin was discovered over two
decades ago and has demonstrated an important biological role in synaptic plasticity,
and associated disorders, a further understanding may help elucidate a more specific
role in these disorders.

CDPPB provided a major breakthrough as the first mGluR5 PAM suitable for in vivo use,
capable of potentiating NMDA receptor currents and exhibiting efficacy in attenuating
schizophrenia relevant behaviours in rodent models. Whilst preclinical investigation of
CDPPB and mGluR5 PAMs is still ongoing, evidence suggests some mGluR5 PAMs,

Jeremy Lum 141

including CDPPB, possess agonist-like activity, subsequently inducing adverse effects
such as seizures and cell death that may be associated with excessive NMDA receptor
activation (Kinney et al., 2005; Parmentier-Batteur et al., 2014). Whilst it was previously
believed NMDA receptor potentiation was vital for the efficacy of mGluR5 PAMs, a new
light has been shed on mGluR5 PAMs, with the development of VU0409551 from Jeff
Conn and colleagues (Rook et al., 2015). VU0409551 is reported to produce
antipsychotic-like and cognitive enhancing effects, via potentiation of mGluR5
downstream signalling, but does not modulate NMDA receptor activity (Balu et al., 2016;
Ghoshal et al., 2017; Rook et al., 2015). Furthermore, it does not produce cell death or
seizure activity even at doses 100x higher than necessary to produce behavioural
efficacy (Rook et al., 2015). VU0409951 is believed to produce a downstream signalling
profile biased from previous mGluR5 PAMs (Rook et al., 2015). The work investigating
VU0409551 is still in its infancy and the molecular mechanisms underlying it behavioural
efficacy are not fully characterised, however provides promise for the continual
development of mGluR5 PAMs, void of neurotoxic side effects and improved clinical
suitability.

Based on the promising behavioural evidence of mGluR5 PAMs, Roche recently

conducted the first human safety and tolerability study for an mGluR5 PAM, RG7342. In
a randomised, double-blind, adaptive study, 37 healthy male subjects were
administered a single dose of RG7342 (ranging from 0.06-1.2 mg; oral) or placebo (Sturm
et al., 2018). RG7342 was tolerated up to 0.6 (under fasted conditions) and 0.9 mg
(under fed condition) in healthy subjects. However, a majority of subjects reported
nausea and dizziness, which were associated with RG7342 plasma concentration.
Furthermore, the estimated terminal half-life of RG7342 was >1000 hours, causing the
investigators to predict it would take >4 months for RG7342 to reach a steady state
concentration. Due to the severity of the RG7342 related symptoms and the
unfavourable half-life of RG7342, the Roche investigators discontinued the
development of RG7342. This initial study does not bode well for the clinical
development of mGluR5 PAMs. However, the fact mGluR5 PAMs display promising
preclinical efficacy in the treatment of all symptoms domains of schizophrenia (Vinson

Jeremy Lum 142

and Conn, 2012) continue to encourage the field to endure efforts to design and
synthesis mGluR5 PAMs with improved in vivo suitability to continue their clinical
development.

6.4 Conclusion
The present body of work provides original findings and further insight into the
glutamatergic system, in particular, group 1 mGluRs in the pathology, development and
treatment of schizophrenia. This work has built from human neurobiology and
progressed to neurodevelopmental, neuropharmacology and culminated in a novel
treatment strategy for schizophrenia, using a rodent model. The findings from
postmortem human brain reveal no involvement, at least using the present measures,
of the glutamatergic system in the nucleus accumbens in schizophrenia. Whilst further
studies using more functional approaches are warranted (where possible in this type of
tissue), the approach used in this thesis to examine the dimeric forms is an important
step to understand the functional properties of group 1 mGluRs and their possible
involvement in schizophrenia. Employing this approach throughout this thesis identified
the unusually high monomeric expression at the juvenile age period, which may have
implications for future therapeutic strategies for mGluR5 PAMs. Finally, this thesis
indicates adolescent treatment with the mGluR5 PAM, CDPPB produces brain-region
specific effects on the glutamatergic system, providing insight into the neurochemical
changes which may underlie its behavioural efficacy. Collectively, this thesis adds to the
current body of research investigating the role of group I mGluRs in the pathology,
current and potentially future treatment of schizophrenia. Unravelling the
neurochemical changes that do or do not characterise schizophrenia and those
important for therapeutic efficacy, are essential in developing our understanding of
schizophrenia pathology and will consequently improve treatment approaches.

Jeremy Lum 143

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