Metabotropic glutamate receptors in
oligodendrocytes and astrocytes in
white and grey matter brain regions
Ilaria Vanzulli
This thesis is submitted in partial fulfillment of the requirements of the award of the degree of Doctor of Philosophy of the University of Portsmouth
University of Portsmouth School of Pharmacy and Biomedical Sciences
September 2014 Abstract
Glial cells express a wide range of neurotransmitter receptors throughout the central nervous system (CNS). In this thesis, I have investigated glial neurotransmitter signaling mechanisms in CNS white matter, using the mouse optic nerve as a model white matter and with a particular focus on metabotropic glutamate receptors (mGluR). The transcriptomic profiling of postnatal, adult and ageing optic nerve identified a pre-eminence of glutamatergic neurotransmission in the optic nerve and highlighted a switch to GABAergic signaling in the ageing white matter, without a loss of glutamatergic neurotransmission.
Immunostaining showed expression of group I and group II mGluR in optic nerve astrocytes and oligodendrocytes and their functionality was demonstrated by live cell calcium imaging. Activation of group I and group II mGluR is shown to protect both oligodendrocytes and astrocytes from hypoxia/ischaemia, using an oxygen and glucose deprivation (OGD) model in the isolated intact mouse optic nerve. A key finding is that mGluR were expressed by glia at all ages and were not developmentally downregulated, as had been suggested from previous in vitro studies. In addition, the genomic analysis indicated disruption of OPCs in ageing tissue, and significant age-related disruption of OPCs was confirmed in the triple transgenic mouse model of Alzheimer’s disease (3xTg AD). This study provides new insight into mGluR expression and function in white matter glia.
I TABLE OF CONTENTS
Abstract I Table of contents II A – List of Tables VIII B – List of Figures X C – Declaration XVI D – Acknowledgements XVII E – Dissemination XVIII F – Glossary of Terms XIX
Section 1 - General introduction 1 1.1 CNS glial cells 2 1.1.1 General features of astrocytes 3 1.1.2 General features of oligodendrocytes 9 1.1.3 Adult OPCs 11 1.2 Myelination 11 1.3 Neurotransmitter receptors in glial cells 14 1.4 The glutamatergic system 17 1.4.1 Metabotropic glutamate receptors 19 1.5 Hypoxia/ischaemia and glutamate excitotoxicity 27 1.6 The optic nerve as a model white matter 30 1.7 Hypothesis and aims 33
Section 2 - Materials and methods 35 2.1 Animals 36
2.1.1 Declaration 36
2.1.2 Animal Strains 36
2.1.3 Animal Ages 37
2.1.4 Tissue Fixation Protocols 38 II 2.2 Molecular Biology 38
2.2.1 Total RNA Extraction 38
2.2.2 Quantitative real time PCR (qRT-PCR) 41
2.2.3 Microarray 42
2.3 Cell Culture 43
2.3.1 Cover Slip Preparation 43 2.3.2 Optic Nerve Explant Cultures 43
2.4 Immunostaining techniques 44
2.4.1 Antibodies 44
2.4.2 Immunocytochemistry 46
2.4.3 Immunohistochemistry 47
2.4.4 Confocal Microscopy 49
2.4.5 Immunohistochemistry negative controls 49
2.4.6 Morphological analysis of OPCs 52
2.4.7 Statistical analysis 52
2.5 Calcium Imaging 53
2.5.1 Optic nerve dissection and dye loading 53
2.5.2 Imaging parameters 53
2.5.3 Typical experiment 54
2.5.4 Solutions 54
2.5.5 Statistical analysis 55
2.6 Oxygen and Glucose Deprivation (OGD) 55
III 2.6.1 Quantification of glia 58
Section 3 - Microarray Analysis of Age-Related Changes in Neurotransmitter Signaling Mechanisms in the Mouse Optic Nerve 59
3.1 Introduction and aim 60
3.2 Results 62
3.2.1 Microarray analysis of gene expression in mouse
optic nerve 62
3.2.2 Macroglial genes expression in optic nerve samples 65
3.2.3 Most expressed genes by glial cells throughout aging 71 3.2.4 Most prominent genes enriched in the P10-12 mouse optic nerve 80 3.2.5 Most prominent genes enriched in the 1-month mouse optic nerve 83 3.2.6 Most prominent genes enriched in the 18-month mouse optic nerve 85 3.2.7 Genes enriched in more than one age group 87 3.2.8 “Neurotransmission machinery” in young, adult and old optic nerve 91 3.2.9 Class A G-protein coupled receptors (GPCRA) in the mouse optic nerve 93 3.2.10 Class C G -protein-coupled receptors (GPCRC) in the mouse optic nerve 96 3.2.11 Expression of ligand-gated ion channels in the mouse optic nerve 98 3.2.12 Expression of genes involved in neurotransmission in the mouse optic nerve 101
3.3 Discussion 106 IV 3.3.1 Age-dependent changes in glial genes 109
3.3.2 Microglial genes and ageing mechanisms in the optic 112
nerve 3.3.3 Age-dependent changes in neurotransmitter receptors in the optic nerve 114
3.3.4 Axonal genes and synaptic release mechanisms in the
optic nerve 118
3.3.5 Astrocytes as a source of synaptic neurotransmitter release
in the optic nerve 120 3.4 Summary and conclusions 121
Section 4 - Functional expression of metabotropic glutamate receptors in the mouse optic nerve 123 4.1 Introduction and aims 124 4.2 Results 126
4.2.1 qRT-PCR analysis of mGluRs in mouse optic nerve
and brain 126 4.2.2 Group I and II mGluRs are expressed in the grey and white matter of the mouse brain 129 4.2.3 Group I and II mGluR expression in mouse optic nerve glia 134 4.2.4 Calcium imaging of mGluR function in optic nerve glia 137 2+ 4.2.5 Group ImGluR mediated raised [Ca ]i in optic nerve glia 141 4.3 Discussion 144 4.3.1 qRT-PCR limitations 144
4.3.2 mGluR5 and mGluR2/3 expression in the mouse
optic nerve 146 4.3.3 mGluR at the gliovascular interface 148 V 4.3.4 mGluR mediate calcium signals in the postnatal mouse
optic nerve glia 149
4.4 Summary and conclusions 151
Section 5 - A protective role for glial metabotropic glutamate receptors in ischaemia 153 5.1 Introduction and aims 154 5.2 Results 156
5.2.1 Group I/II mGluR activation protects optic nerve
cells from ischaemia 156 5.2.2 Astrocytes in the postnatal optic nerve are susceptible to OGD and are protected by mGluR activation 159 5.2.3 Oligodendrocytes in the postnatal optic nerve are susceptible to OGD and are protected by mGluR activation 161 5.2.4 Oligodendrocytes in the adult optic nerve are susceptible to OGD and are protected by mGluR activation 164 5.2.5 The protective effect of goup I mGluR on postnatal optic nerve oligodendrocytes in OGD requires PKC activation 166 5.2.6 Gq coupled receptors are protective against OGD in postnatal optic nerve oligodendrocytes 168 5.2.7 Role of adenylate cyclase in the protective effect of group II mGluR on postnatal optic nerve oligodendrocytes in OGD 171 5.3 Discussion 173
5.3.1 Hypoxia/Ischemia effects on astrocytes 174
5.3.2 Hypoxia/Ischaemia effects on oligodendrocytes 177
5.4 Summary and Conclusions 180
VI Section 6 - Changes in Oligodendroglial Cells in the Mouse 3xTg Model of Alzheimer’s Disease 181 6.1 Introduction and aims 182 6.2 Results 184
6.2.1 mGluR5 expression in the ageing and 3xTg-AD
hippocampus 184 6.2.2 Age-related changes in OPCs in the hippocampus in 3xTg-AD and age-matched controls 187 6.2.3 Age-related changes in myelination in 3xTg-AD hippocampus and age-matched controls 194 6.3 Discussion 197 6.3.1 The 3xTg-AD model 197 6.3.2 Changes in OPCs and impact on myelination in 3xTg-AD 198 6.4 Summary and conclusions 201
Section 7 - General discussion 203 7.1 Summary of key findings 204 7.2 Glutamatergic neurotransmission mechanisms in white matter 205 7.3 Purinergic neurotransmission in white matter 208 7.4 Altered GABAergic and Glutamatergic neurotransmission in ageing white matter 210 7.5 mGluRs are protective for white matter 211
7.6 Link between ageing and hypoxia 213
7.7 Myelination and oligodendrocytes in ageing and pathology 215 7.8 Conclusions 217
Section 8 – Appendix 218 References 223
VII A - List of tables
Section 1
Table 1.1 Major functions of astrocytes and references 7
Table 1.2 Differential expression of ionotropic glutamate receptors in astrocytes, oligodendrocytes and OPCs 19
Table 1.3 Group classification of metabotropic glutamate receptors 21
Section 2
Table 2.1 Primary antibodies 45
Table 2.2 Secondary antibodies 45
Table 2.3 Confocal microscope characteristics 49
Table 2.4 List of pharmacological agents used in calcium imaging 55
Table 2.5 List of pharmacological agents used in OGD 57
Section 3
Table 3.1 Gene expression levels of well described cell markers for astrocytes, oligodendrocytes, OPCs, neurons, microglia and endothelial cells 69
Table 3.2 Expression levels of the top 100 genes in P10-12 optic nerves 73
Table 3.3 Expression levels of the top 100 genes in 1-month optic nerves 75
Table 3.4 Expression levels of the top 100 genes in 18-month optic nerves 77 VIII Table 3.5 Most prominent genes enriched in the P10-12 mouse optic nerve 82
Table 3.6 Most prominent genes enriched in the 1-month mouse optic
Nerve 84
Table 3.7 Most prominent genes enriched in the 18-month mouse optic nerve 86
Table 3.8 Enriched genes common to P10-12 and 1-month mouse optic nerves 88
Table 3.9 Enriched genes common to P10-12 and 18-month mouse optic nerves 89
Table 3.10 Enriched genes common to 1-month and 18-month mouse optic nerves 89
Table 3.11 Enriched genes common to P10-12, 1-month and 18-month mouse optic nerves 90
Table 3.12 GPCRA in P10-12, 1-month and 18-month mouse optic nerve 94
Table 3.13 GPCRC in P10-12, 1-month and 18-month mouse optic nerve 97
Table 3.14 LGIC in P10-12, 1-month and 18-month mouse optic nerve 99
IX B - List of figures
Section 1
Figure 1.1 Morphological diversity of astrocytes throughout the CNS 4
Figure 1.2 CNS astrocytes in the GFAP-EGFP reporter mouse 5
Figure 1.3 Mechanisms of intercellular Ca2+ signaling in astrocytes 8
Figure 1.4 Myelinating oligodendrocyte 9
Figure 1.5 Markers of oligodendrocyte lineage specification and maturation 11
Figure 1.6 Oligodendrocyte progenitor cell (OPC) in the adult brain 12
Figure 1.7 Neurotransmitter receptors in glial cells 15
Figure 1.8. Astroglial glutamate receptors in brain metabolism 16
Figure 1.9. Neurotransmission and oligodendrocyte differentiation 17
Figure 1.10 Generic mGluR structure 21
Figure 1.11 Distribution of mGluR subunits in the mouse brain 23
Figure 1.12 Distribution and synaptic modulatory function of mGluRs in excitatory synapses 24
Figure 1.13 Cytoprotective effects achieved through mGluRs activation 26
Figure 1.14 Mechanisms of glutamate excitotoxicity 29
Figure 1.15 Diversity of neurotransmitters in the optic nerve 31
Section 2
Figure 2.1 Negative controls for immunohistochemistry 51
X Section 3
Figure 3.1 Gene expression analysis of P10-12, 1-month and 18-month mouse optic nerve 64
Figure 3.2 Gene expression analysis of well described cell specific marker in P10-
12, 1-month and 18-month mouse optic nerve 68
Figure 3.3 Age-related changes in the proportional expression of markers for astrocytes, oligodendrocytes, OPCs, axons, microglia and endothelial cells in mouse optic nerve 70
Figure 3.4 Venn diagram illustrating the distribution of the top 100 most expressed entities in P10-12, 1-month and 18-month optic nerve 72
Figure 3.5 Top 100 most expressed entities in P10-12, 1-month and 18-month mouse optic nerve, classified according to their coding protein biological function and expressed as percentage 79
Figure 3.6 Venn diagram showing the number of entities for neurotransmission in filtered for E50 and FC 1.5 between at least 2 age groups 92
Figure 3.7 Age-related changes of the most prominent GPCRA in the mouse optic nerve 95
Figure 3.8 Age-related changes of the most prominent GPCRA in the mouse optic nerve 97
Figure 3.9 Age-related changes of the most prominent LGIC in the mouse optic nerve 100
Figure 3.10 Age-related changes of the most prominent neurotransmitter transporters in the mouse optic nerve 103
XI Figure 3.11 Age-related changes of the most prominent enzymes involved in neurotransmission in the mouse optic nerve 104
Figure 3.12 Age-related changes of the most prominent SNARE and synaptic proteins involved in neurotransmission in the mouse optic nerve 105
Figure 3.13 Summary of age-related changes in the main neurotransmitter receptors in the mouse optic nerve 108
Figure 3.14 Age-related changes in the main oligodendrocyte and OPC genes in the mouse optic nerve 111
Figure 3.15 Age-related changes in the neurotransmitter receptor expression in the mouse optic nerve 116
Section 4
Figure 4.1 qRT-PCR melt curve analysis 127
Figure 4.2 Expression of mGluR transcripts in cortex, cerebellum and optic nerve of P9, P12 and P30 mice 128
Figure 4.3 mGluR5 expression in grey and white matter of the mouse forebrain 131
Figure 4.4 mGluR2/3 expression in grey and white matter of the mouse forebrain 132
Figure 4.5 mGluR5 and mGluR2/3 colocalisation with astrocytes, pericytes and
NG2 cells in grey matter of the mouse forebrain 133
Figure 4.6 mGluR5 and mGluR2/3 immunolabelling in the mouse optic nerve 135
Figure 4.7 mGluR5 and mGluR2/3 immunolabelling in optic nerve glial
XII cultures 136
Figure 4.8 Comparison of response to ATP, glutamate and ACPD in optic nerve glia 139
Figure 4.9 Calcium responses in optic nerve glia in response to the group I/II agonist ACPD 140
Figure 4.10 Group I mGluR mediate calcium signals in optic nerve glia 142
Figure 4.11 Group I mGluR blockade significantly decreases the response to ACPD 143
Section 5
Figure 5.1 Effect of the Group I/II mGluR agonist ACPD on OGD induced cell death in postnatal mouse optic nerve 158
Figure 5.2 mGluR activation protects postnatal optic nerve astrocytes from OGD 160
Figure 5.3 mGluR activation protects postnatal optic nerve oligodendrocytes from OGD 162
Figure 5.4 mGluR activation protects postnatal optic nerve mature oligodendrocytes from OGD 163
Figure 5.5 mGluR activation protects adult optic nerve oligodendrocytes from
OGD 165
Figure 5.6 Protective effect of group I mGluR on postnatal optic nerve oligodendrocytes in OGD requires PKC activation 167
Figure 5.7 Activation of the Gq-coupled P2Y1 receptor protects postnatal optic nerve oligodendrocytes from OGD 169
XIII Figure 5.8 dbcAMP blocks the protective effect of group II mGluR on postnatal optic nerve oligodendrocytes in OGD 171
Figure 5.9 Forskolin blocks the protective effect of group II mGluR on postnatal optic nerve oligodendrocytes in OGD 172
Section 6
Figure 6.1 mGluR5 in the 3xTg-AD mouse hippocampus and age-matched controls 186
Figure 6.2 Distribution and morphology of OPCs in the hippocampus of 6-month non-Tg mouse 190
Figure 6.3 OPC ‘sister cells’ in the hippocampus of 6-month non-Tg mouse 191
Figure 6.4 Age-dependent changes in OPC sister cells in 3xTg-AD
Hippocampus 192
Figure 6.5 Morphological analysis of OPCs in the 3xTg-AD hippocampus and age- matched non-Tg controls 193
Figure 6.6 Decrease of MBP immunostaining in the 3xTg-AD
Hippocampus 196
Section 7
Figure 7.1 Neurotransmitter profile of the optic nerve 205
Figure 7.2 Age-dependent changes in vesicular apparatus in the optic nerve 208
XIV Section 8
Figure 8.1 Relationship between OPCs and A in 3xTg-AD 219
Figure 8.2 Relationships between OPCs and A plaques in the hippocampus of
24-month 3xTg-AD 220
Figure 8.3 Astrocytes distribution and morphology in the hippocampus of 6- month non-Tg control 221
Figure 8.4 Interrelations between OPCs and astrocytes with A plaques in the 24- month 3xTg-AD hippocampus 222
XV C – Declaration
Whilst registered as a candidate for the above degree, I have not been registered for any other research award. The results and conclusions embodied in this thesis are the work of the named candidate and have not been submitted for any other academic award.
XVI D – Acknowledgements
First and foremost I would like to thank my supervisor Prof Arthur Butt for his guidance and support from day one to the submission date.
This work is dedicated to my mother and my brother; they have always been and they will always be there for me, no matter how far away that “there” is geographically, their love and support is bigger than any distance.
My most heartfelt thanks go to my colleagues and friends Maria, Andrea,
Francesca, James, Ginny, Federica, Gloria and Sam. We worked, discussed, drunk, danced and laughed a lot together. I found a second family here and thanks to you I have another place to call home. You are my ohana and I’ll always have you under my skin.
Finally, I would like to thank the Marie Curie ITN EduGlia programme for funding my PhD and to all the members of the network for the many days filled with science and fun spent together.
XVII E – Dissemination
8th IBRO World Congress of Neuroscience, July 2011, Florence, IT – poster presentation “Metabotropic glutamate receptors mediate Calcium signals in CNS white matter glia”
10th Euroglia Meeting on Glial Cells in Health and Diseases, September 2011, Prague, CZ – poster presentation “Adrenergic and metabotropic glutamate receptors mediate calcium signals in CNS white matter glia”
Society for Neuroscience 42nd Annual Meeting, October 2012, New Orleans, US – poster presentation “Functional expression of metabotropic glutamate receptors in optic nerve glia”
XI European Meeting on Glial Cells in Health and Disease, July 2013, Berlin, DE – poster presentation “Glial expression of metabotropic glutamate receptors in the mouse optic nerve: a role in ischaemia mediated disruption in postnatal CNS white matter”
XVIII F – Glossary of terms
A = Amyloid-beta aCSF = Artificial Cerebrospinal Fluid
AD = Alzheimer’disease
ALS = Amyotrophic lateral sclerosis
ANOVA = Analysis of Variance
ATP = Adenosine triphosphate
BBB = Blood Brain Barrier
BSA = Bovine Serum Albumine
CA1 = Cornus Ammonis 1
CA3 = Cornus Ammonis 3
CSF = Cerebrospinal Fluid
CNP = 2’,3’-Cyclic Nucleotide 3’-phosphodiesterase
CNS = Central Nervous System
CSPG = Chondroitin Sulphate Proteoglycan
DAG = Dyacylglycerol
DG = Dentate Gyrus
DIV = Days in vitro
DsRed = Discosoma sp. Red
EAAT = Excitatory Amino Acid Transporter
EAE = Experimental Autoimmune Encephalomyelitis
EGFP = Enhanced Green Fluorescent Protein
FOV = Field of View
GFAP = Glial Fibrillary Acidic Protein
XIX GPCR = G-protein Coupled Receptor
Kir = Inward Rectifying Potassium Channel
MBP = Myelin Basic Protein mGluR = Metabotropic Glutamate Receptor
MS = Multiple Sclerosis
NG2 = Neuron-glial 2
NTG = Non Transgenic
OL = Oligodendrocytes
ON = Optic nerve
OPC = Oligodendrocyte Progenitor Cell
P = Postnatal Day
PB = Phosphate Buffer
PBS = Phosphate Buffer Saline
PD = Parkinson’s Disease
PKC = Protein kinase C
PLC = Proteolipase C
PLP = Proteolipid Protein
PVL = Periventricular Leukomalacia
RT = Room Temperature
SEM = Standard Error of the Mean
TB = Trizma Buffer
XX
Section 1
General Introduction
1 1.1 CNS glial cells
The central nervous system (CNS) is composed of two main cell types – neurones and neuroglia. The term ‘neuroglia’ was coined in 1856 by Rudolf
Ludwig Karl Virchow, who described it as ‘nerve glue’, the mere connective tissue of the brain (comprehensively reviewed in Verkhratsky and Butt, 2013). In the following decades glial cells became the interest of the Nobel prize winning histologists Camillo Golgi and Santiago Ramon y Cajal, who clearly described the morphology of astrocytes using metal impregnation staining techniques. It was
Cajal’s student, Pio Del Rio Hortega, who developed the metal staining technique further to provide the first descriptions of oligodendrocytes and microglia. Nowadays, CNS glia are broadly classified into astrocytes, oligodendrocytes, ependymal cells and microglia, in addition to a population of oligodendrocyte progenitor cells (OPCs), or NG2-glia, that populate the adult brain. The two main macroglial cell types, astrocytes and oligodendrocytes, have the same lineage as neurones and are derived from embryonic neural stem cells with a neuroectodermal origin. Ependymal cells are also neuroectodermal and line the ventricles of the brain and central canal of the spinal cord, where they assist in the production, monitoring and circulation of the cerebrospinal fluid (CSF). Microglia are not neural cells: they are derived from the yolk sac macrophages that seed the neuroepithelium during early fetal development. Embryonic microglia expand and colonise the whole CNS until adulthood, to serve as the resident macrophages and immune-competent cells of the so-called ‘immune privileged’ CNS (Ginhoux et al., 2013).
2 1.1.1 General features of astrocytes
Astrocytes, or astroglia, in general are star-shaped cells that can be identified by their expression of the intermediate filament glial fibrillary acidic protein (GFAP), the metabolic enzyme glutamine synthetase (GS) or the glutamate transporters GLAST and GLT-1. Astrocytes are broadly divided into two main subtypes according to their morphology and location - protoplasmic astrocytes of the grey matter and fibrous astrocytes of the white matter (Figure
1.1). However, these are umbrella terms, and astrocytes display a very wide range of morphologies and highly varied expression of GFAP, in the rodent CNS
(Figure 1.1) (Emsley & Macklis, 2006) and even more so in the human brain
(Oberheim et al., 2012). One of the most established methods for identifying astrocytes is the use of transgenic reporter mice, in which GFAP drives the expression of a fluorescent reporter, such as enhanced green fluorescent protein (EGFP)(Nolte et al., 2001), as illustrated in Figure 1.2.
3
Figure 1.1 Morphological diversity of astrocytes throughout the CNS. Representative astrocytes from different regions of the brain and spinal cord of an adult transgenic mouse expressing enhanced green fluorescence protein under control of the GFAP astrocyte specific promoter (hGFAP-EGFP) (Emsley & Macklis, 2006).
4
Figure 1.2 CNS astrocytes in the GFAP-EGFP reporter mouse (Robel et al., 2011)
Protoplasmic astrocytes are expressed throughout the grey matter and have highly branched fine processes that contact the basement membrane surrounding the blood vessels and enwrap synaptic connections (Figure 1.2a).
White matter fibrous astrocytes have thicker and less branched processes that contact axons at nodes of Ranvier (Figure 1.2b). In addition, other astrocytes have a radial morphology and include the Bergmann glia in the cerebellum and the Müller glia in the retina. In the CNS there is no region devoid of astrocytes; which are organized in a contiguous, non-overlapping manner (Halassa et al.,
2007).
Astrocytes perform a wide range of functions that are essential for CNS functioning and integrity (Table 1.1) (Verkhratsky and Butt, 2013). As well as providing the physical support and structure of the CNS, astrocytes provide for homeostasis of the extracellular environment, primarily through uptake of K+ and glutamate, which are constantly released by neurones and have pathological consequences if allowed to build up (Martinez-Hernandez et al.,
1977; Kimelberg et al., 1979; Levi et al., 1983; Ransom & Sontheimer, 1992;
5 Horio, 2001; Kofuji & Newman, 2004). Astrocytes also provide energy substrates to neurones (Pellerin & Magistretti, 1996; Suh et al., 2007), and help couple cerebral blood flow to neuronal activity (Iadecola & Nedergaard, 2007; Koehler et al., 2009). In addition, astrocytes express neurotransmitter receptors and are able to release neurotransmitters, and can directly modulate synaptic transmission (Haydon, 2001; Allen & Barres, 2005). Astrocytes play an important role in brain development, by providing a scaffold along which neurones can migrate and extend their axons (Powell & Geller, 1999), strengthening newly formed synapses (Ullian et al., 2001), regulating neurogenesis and gliogenesis (Doetsch, 2003), and playing an essential role in development and maintenance of the blood-brain barrier (BBB) (Araya et al.,
2008). Furthermore, astrocytes play a key role in CNS defence and neuroprotection, undergoing ‘reactive astrogliosis’ in response to CNS insults, characterised by formation of a ‘glial scar’, together with immune responses and secretion of inflammatory factors (Sofroniew, 2009). Thus, astrocytes can be described as homeostatic cells and their dysfunction can have dire consequences for neuronal function and integrity, and hence astrocytes are central to all neuropathologies (Verkhratsky and Butt, 2013).
6 Table 1.1 Major functions of astrocytes and references
Regulation of extracellular environment: (Kimelberg et al., 1979) Homeostasis of H+ (Ransom & Sontheimer, 1992) K+ buffering (Martinez-Hernandez et al., 1977) Glutamate and GABA uptake (Levi et al., 1983) Support for neurons through the (Pellerin & Magistretti, 1996) astrocyte-neuronal lactate shuttle Control of cerebral blood flow and (Zonta et al., 2003b) functional hyperaemia Modulation of synaptic transmission (Haydon, 2001) Regulation of brain development: (Powell & Geller, 1999) Neuronal migration (Ullian et al., 2001) Synapse formation (Doetsch, 2003) Neurogenesis and gliogenesis Formation and maintenance of the blood (Araya et al., 2008) brain barrier Reaction to CNS injuries (reactive gliosis) (Sofroniew, 2009)
A defining physiological feature of astrocytes is that they are non- excitable, i.e. they do not conduct action potentials as do neurones. However, astrocytes express voltage-gate sodium channels (Nav) and both voltage-gated potassium channels (Kv) and inward rectifying potassium channels (Kir) that carry inward and outward currents in response to shifts in the membrane potential, which in neurones is the molecular and ionic basis of the action potential (Nedergaard et al., 2003). Moreover, astrocytes express a very wide- range of neurotransmitter receptors that on activation result in changes in the membrane potential, but unlike neurones these do not trigger action potentials because the density of Nav is too low and the density of Kv and Kir is very high in
7 the astrocyte cell membrane, essentially clamping the astrocyte cell membrane close to the equilibrium potential for potassium (EK) (Kimelberg et al., 2006).
Instead of electrical excitability, activation of astrocytes display regulated
2+ increases in intracellular calcium concentration ([Ca ]i) in response to receptor activation (Figure 1.3), and this represents astroglial ‘calcium excitability’ and their mechanism of intracellular and intercellular communication (Cornell-Bell et al., 1990; Charles et al., 1991).
Figure 1.3 Mechanisms of intercellular Ca2+ signaling in astrocytes.
2+ Intercellular Ca signals can be propagated by the passage of IP3 through gap junctions (A), or release of neurotransmitters such as ATP (B), or a combination of both (adapted from Verkhrtasky and Butt, 2013)
8 1.1.2 General features of oligodendrocytes
The main function of oligodendrocytes is the formation of myelin, the fatty multilamellar structure that insulates axons and guarantees high-speed conduction of action potentials. Each oligodendrocyte can myelinate up to 30 axons within a short distance of the cell body (Butt & Ransom, 1989; Ransom et al., 1991) (Figure 1.4). Failure in myelin formation (dysmyelination) and myelin degradation (demyelination) affect neuronal activity; both of these processes are important in neuropathology, as observed in demyelinating diseases such as multiple sclerosis (MS) and leukodystrophies, as well as neurodegenerative diseases, such as Alzheimer’s disease, where myelin loss has been described
(Bartzokis et al., 2003).
Figure 1.4 Myelinating oligodendrocyte (Lundgaard et al., 2013)
9 Oligodendrocytes in the forebrain are derived from oligodendrocyte progenitor cells (OPCs) that originate from neural stem cells (NSC) of the subventricular zone (SVZ) (Richardson et al., 2006). During development, OPCs migrate to their destinations in the brain, where they proliferate and differentiate into oligodendrocytes in response to endogenous mechanisms and local environmental cues. OPCs are defined by their expression of the chondrotin sulphate proteoglycan (CSPG) neuron-glia 2 (NG2) and of the platelet-derived growth factor receptors of the subtype (PDGFR) (Mudhar et al., 1993;
Nishiyama et al., 1996a; b; Butt et al., 1997a; Butt et al., 1997b). The differentiation of OPCs into oligodendrocytes is characterized by a number of key stages that can be distinguished by expression of stage-specific transcription factors and myelin-related proteins (Figure 1.5); key stages are that once OPCs stop proliferating and begin to differentiate into oligodendrocytes they lose NG2 and PDGFR, and first express early markers such as galactocerebroside (GalC), and then the myelin-specific proteins myelin basic protein (MBP) and proteolipid protein (PLP). Proliferation and differentiation of OPCs is controlled by a large number of factors, some of the most important being PDGF, fibroblast growth factor (FGF2), Wnt signalling, insulin-like growth factor (IGF-I) and Sonic hedgehog (Chandran et al., 2003;
Fancy et al., 2009; Kim et al., 2009; Azim & Butt, 2011).
10
Figure 1.5 Markers of oligodendrocyte lineage specification and maturation (Schumacher et al., 2012)
1.1.3 Adult OPCs
A substantial population of OPCs persists in the adult CNS after development and the main period of oligodendrocyte generation and myelination (Figure 1.6); adult OPCs have also been termed NG2-glia, since they are primarily identified by their expression of this CSPG, but NG2 is also expressed by pericytes in the CNS, and OPCs can be distinguished by their co- expression of the classically defined OPC markers PDGFR and O4 (Nishiyama et al., 1996a; Reynolds & Hardy, 1997). Adult OPCs are found throughout the grey and white matter and form synaptic contacts with neurones and axons at nodes of Ranvier (Bergles et al., 2000; Butt et al., 2005; Kukley et al., 2007; Ziskin et al.,
2007). Adult OPCs generate new oligodendrocytes at a slow rate throughout life and this is increased following CNS injury and/or demyelination (Dimou et al.,
2008; Rivers et al., 2008; Zhu et al., 2008b; Kang et al., 2010). Adult OPCs
11 represent the largest proliferative cell population in the adult brain (Dawson et al., 2003; Butt et al., 2005), although their cell cycle time gets longer with ageing (Psachoulia et al., 2009; Simon et al., 2011). Like stem cells, OPCs divide asymmetrically to form ‘sister OPCs’, one for self-renewal of the OPC population and the other differentiates into an oligodendrocyte. OPCs are vital for restoring myelin, by proliferating in the injury site and giving rise to new oligodendrocytes
(Tripathi et al., 2010; Guo et al., 2011).
Figure 1.6 Oligodendrocyte progenitor cell (OPC) in the adult brain (Vanzulli, unpublished)
1.2 Myelination
Myelin is an electrical insulator composed of 70% lipids and 30% proteins.
The most abundant proteins, and therefore myelin markers, are proteolipid protein (PLP), the myelin basic protein (MBP), the myelin oligodendrocyte
12 glycoprotein (MOG), the myelin associated glycoprotein (MAG) and the 2’, 3’- cyclic nucleotide 3’-phosphodiesterase (CNPase) (Dubois-Dalcq et al., 1986). In the mouse brain, myelination starts at birth in the hindbrain and proceeds caudally and rostrally (Foran & Peterson, 1992; Baumann & Pham-Dinh, 2001).
Myelination in vivo requires neuron-glia recognition that can be organized in subsequent stages (Butt & Berry, 2000):
Contact between axon and immature oligodendrocyte
Induction phase: OPC extends initiator processes along a receptive axon
Remodeling phase: oligodendrocyte loses non-myelinating processes
and myelin wraps around the axon initiate with the formation of
internodal segments and the establishments of nodes of Ranvier
Maturation phase: myelin sheaths grow to establish the adult axonal
diameter and myelin is compacted
Several molecules control this process: integrins and adhesion molecules are believed to be involved in the initiation of myelination (Laursen & Ffrench-
Constant, 2007). Neuregulin is thought to be responsible for the decision of an oligodendrocyte to myelinate a particular axon (Sherman & Brophy, 2005), whereas Fyn kinase has been reported to promote oligodendroglial process growth and to initiate myelin wrapping (Klein et al., 2002).
Myelination is not a process restricted to the postnatal age. White matter volume keeps increasing until the fourth decade of life in humans
(Bartzokis et al., 2001) and around 70% of corpus callosum is still non- myelinated in eight month-old mice (Sturrock, 1980). These evidences and the 13 existence of a pool of adult OPCs indicate that myelination is an active and plastic process throughout life (Rivers et al., 2008). The ‘myelin model’ proposes that myelin is the basis to vertebrate cognitive function and that in humans developmental deficits in myelin as well as injury or age-related breakdown and loss of myelin are a fundamental substance of psychiatric and neurodegenerative disorders, including schizophrenia, bipolar disease and
Alzheimer’s disease (AD) (Bartzokis, 2002; Bartzokis et al., 2003; Bartzokis,
2004; Bartzokis et al., 2007; Bartzokis, 2011a). It is known that CNS plasticity and repair decreases with age, and there is an age-related decline in the capacity for OPCs to regenerate new oligodendrocytes (Rivers et al., 2008;
Psachoulia et al., 2009). This has led to the hypothesis that failure in recruiting
OPCs and in their differentiation into myelinating oligodendrocytes is a central feature of the failure of myelination in the ageing brain and in demyelinating disease (Shields et al., 1999; Franklin, 2002; Sim et al., 2002).
1.3 Neurotransmitter receptors in glial cells
The purinergic and glutamatergic receptors are the main signaling systems that regulate glial functions, but glial cells express receptors for the main CNS neurotransmitters, neuropeptides, cytokines and chemokines (for detailed list see (Verkhratskiĭ & Butt, 2013)) and can release many signaling molecules
(Figure 1.7).
14
Figure 1.7 Neurotransmitter receptors in glial cells. (Adapted from Verkhrtasky and Butt, 2013)
In astrocytes, neurotransmitter receptors are the sensors by which astrocytes are able to respond to changes in neuronal synaptic signaling, for example activation of glutamate receptors stimulates Na+/K+ pumps, glutamate uptake, and astroglial metabolic support for neurones, by stimulating lactate release and regulating local blood supply (Figure 1.8).
15
Figure 1.8. Neurotransmitters in astrocyte functions. (1) Astrocytes uptake synaptically released glutamate via specific glutamate transporters. (2) Communication between astrocytes occurs via ATP release and binding to purinergic receptors, expressed on adjacent astrocytes, which elicits a downstream calcium mobilization. (3) Gap junctions allow the exchange of small molecules and therefore contribute to cell- cell communication. (4) Astrocytes are responsible for the replenishment of neuronal glutamate via the glutamate-glutamine cycle and (5) for energy supply and storage thanks to their ability to transport glucose from the vasculature. (6) The release of vasoactive substances at the astrocyte end-feet regulates local blood flow. (7) Increase in intracellular calcium concentration might lead to the release of glutamate and other factors, i.e. prostaglandins. Glutamate release through hemichannels can be induced in vitro through lowering of extracellular calcium. (9) Glutamate binding to metabotropic glutamate receptors on astrocytes activates intracellular calcium, leading to the release of vasodilatatory substances. Abbreviations: Gln, glutamine; Glu, glutamate; IP3, inositol trisphosphate; PLC, phospholipase C. (Maragakis & Rothstein, 2006)
Oligodendrocytes and OPCs also express a wide range of ion channels and neurotransmitter receptors, although with the exception of glutamate receptors, these are poorly defined (Verkhratsky and Butt, 2013). A common feature in oligodendrocytes is that neurotransmitters receptors are generally downregulated with differentiation, and numerous in vitro studies provide evidence they can regulate OPC proliferation, migration and differentiation
(Figure 1.9). However, little is known about the physiological functions of 16 oligodendroglial neurotransmitter receptors and evidence they regulate myelination in vivo or in situ is lacking (for recent review, see (Butt et al.,
2014a)).
Figure 1.9. Neurotransmission and oligodendrocyte differentiation. Oligodendrocytes express a number of neurotransmitter receptors that are generally downregulated with age and are implicated in regulation of differentiation and myelination (From Verkhratsky and Butt, 2013).
1.4 The glutamatergic system
A key feature of both astrocytes and oligodendrocytes is their expression of glutamate receptors. Glutamate is the main excitatory neurotransmitter in the mammalian CNS, and plays a crucial role in primary physiological neuronal and glial functions, and it is pivotal for higher brain functions such as learning, memory and cognition (Watkins & Evans, 1981). Furthermore, dysregulation of the glutamatergic signaling and glutamate excitotoxicity have been implicated
17 in an array of neuropathologies, with central roles in stroke and amyotrophic lateral sclerosis (ALS), as well as multiple sclerosis, epilepsy and schizophrenia
(Nakanishi, 1992; 1994; Beneyto et al., 2007). Glutamate receptors are classified in ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). iGluRs are ligand-gated ion channels permeable to K+, Na+ and Ca2+ and are classified into three groups according to the name of the agonist that binds to them with highest specificity, namely NMDA (N-Methyl-D-
Aspartate), AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate), and
Kainate (KA) receptors (Wisden & Seeburg, 1993; Lodge, 2009). AMPA receptors are assembled from four subunits, GluA1-4, all of which have been identified in astrocytes (Seifert & Steinhauser, 2001). The absence of the GluA2 subunit confers greater Ca2+ permeability on the AMPA receptor, which is a feature in
OPCs (Hamilton et al., 2010), and it is suggested that oligodendroglial AMPA receptors comprise exclusively GluA3 and GluA4 (Itoh et al., 2002). Functional
KA receptors have not been detected on astrocytes, but oligodendrocytes express GluK2-5 subunits (De Biase et al., 2010). Until quite recently, glial cells were not considered to express NMDA receptors, but physiological and immunocytochemical studies have identified functional NMDA receptors in astrocytes (Schipke et al., 2001) and oligodendrocytes (Káradóttir et al., 2005).
NMDA receptors assemble as heteromers that require a GluN1 subunit and at least one GluN2 subunits (from GluN2A-D). Astrocyte and oligodendrocyte
NMDA receptors are likely to be formed from two GluN1, one GluN2 and one
GluN3 subunit, which reduces their Mg2+ block and makes them active at resting membrane potential (Káradóttir et al., 2005; Lalo et al., 2011). mRNA for all
18 NMDA receptor subunits have been detected in the optic nerve (Salter & Fern,
2005) (Table 1.2).
Table 1.2 Differential expression of ionotropic glutamate receptors in astrocytes, oligodendrocytes and OPCs.
Astrocytes Oligodendrocytes OPCs
GluN1, 2A-D and Functional expression of GluN1, GluN2C and GluN3A-B tri-heteromers of GluN1, GluN3A/B NMDA (Conti et al., 1996; GluN2C and GluN3A/B (Karadottir et al., 2008; Schipke et al., 2001; Lee (Salter & Fern, 2005; Domingues et al., 2011) et al., 2010) Burzomato et al., 2010)
All four known subunits GluA2, GluA3, GluA4 GluA3 and GluA4. GluA1-4 AMPA (Itoh et al., 2002; De GluA2 non edited or (Seifert & Steinhauser, Biase et al., 2010) absent (Li & Stys, 2000) 2001)
GluK2-5
(Sanchez-Gomez & No functional expression GluK2-5 KA Matute, 1999; De Biase detected yet (Kukley & Dietrich, 2009) et al., 2010)
1.4.1 Metabotropic glutamate receptors
mGluRs are members of the seven transmembrane domain G protein- coupled receptor (GPCR) C family (Figure 1.10) and represented by three groups associated with distinct intracellular pathways. Eight different mGluRs have been cloned and they are divided in three groups, based on their second
19 messenger coupling, pharmacology and amino acid sequence (Pin & Bockaert,
1995). Members of the same group share approximately 70% sequence homology, which is reduced to 45% between members of different groups
(Conn & Pin, 1997). Group I mGluRs (mGluR1 and mGluR5) are positively coupled to phospholipase C (PLC) via Gq/11 and inositol triphosphate (IP3) formation. Their activation leads to an increase in intracellular calcium levels
2+ ([Ca ]i) by inducing calcium release from intracellular stores. Both group II
(mGluR2 and mGluR3) and III (mGluR4, mGluR6, mGluR7 and mGluR8) are negatively coupled to adenylyl cyclase via Gi/G0 (Table 1.3). In addition, activation of group II mGluRs can lead to stimulation of K+ channels, inhibition of voltage-gated Ca2+ channels, and activation of the MAPK and PI3K signaling pathways (Pin & Duvoisin, 1995).
20
Figure 1.10 Generic mGluR structure. A schematic representation of a monomeric form of the metabotropic glutamate receptor; the N-terminal contains the binding site for glutamate and other competitive agonists/antagonists whilst non-competitive antagonists bind to the 7TM domain. (Spooren & Gasparini, 2004)
Table 1.3 Group classification of metabotropic glutamate receptors. From IUPHAR database website
21
In situ hybridization analyses illustrate the expression mGluRs RNA in different brain regions (Figure 1.11). mGluR1 is highly expressed in cerebellum and olfactory bulb (Masu et al., 1991) and its expression is detected also in neuronal cells of the hippocampus and in cerebellar purkinje cells (Masu et al.,
1991), in the substantia nigra, lateral septum, globus pallidum and thalamic relay nuclei (Shigemoto et al., 1992; Baude et al., 1993). mGluR5 RNA has been detected most abundantly in the striatum and lower in the cerebellum and pons; it is expressed in hippocampus, striatum and lateral septal nucleus (Masu et al., 1991; Abe et al., 1992; Shigemoto et al., 1992). The expression of mGluR5 is developmentally downregulated and different splice variants are expressed in different regions and at different time through life: mGlu5a isoform is expressed highly in all brain regions during early postnatal ages, whereas mGlu5b is highly expressed in the hippocampus, striatum and cerebral cortex in adult (Romano et al., 1996). mGluR2 and mGluR3 expression is widespread throughout the CNS
(Nicoletti et al., 2011), highest in the cerebellar cortex and in the olfactory bulb
(Ferraguti & Shigemoto, 2006), whereas group III subunits have more specialized expression, for example mGluR4 is abundant in the parallel fibers of cerebellar granule cells (Kinoshita et al., 1996).
22
Figure 1.11 Distribution of mGluR subunits in the mouse brain. In situ hybridization labeling of midline sagittal brain sections from a P56 male C57BL/6J mouse. OB: olfactory bulb; CTX: cortex; HP: hippocampus; CBL: cerebellum. Images from Allen Brain Atlas.
At the cellular level, group I subunits are localised mainly at the postsynaptic membrane in the vicinity of NMDA and AMPA receptors (Baude et al., 1993; Petralia et al., 1996), whereas group I receptors, group II and III mGluRs are mainly located presynaptically (Ferraguti & Shigemoto, 2006). mGluR act as neuromodulators involving the generation of slow excitatory and inhibitory synaptic potentials, and hence modulation of synaptic transmission, synaptic integration, and plasticity (Figure 1.12) (Coutinho & Knopfel, 2002). mGluR-subtypes are implicated in multiple nervous system disorders, including
23 ischaemia-induced brain damage, traumatic brain injury, Huntington's and
Parkinson's-like pathology, epilepsy, and Fragile-X syndrome (Flor et al., 2002).
Figure 1.12 Distribution and synaptic modulatory function of mGluRs in excitatory synapses. (Benarroch, 2008)
24 Experimental studies have demonstrated that mGluRs can increase cellular survival during injury paradigms through the activation of the PI3-K/Akt pathway (Figure 1.13) (Chong et al., 2003). In rat hippocampal neuronal cultures, application of the group I mGluR agonists prevent neuronal injury during oxidative stress through increased Akt activity (Chong et al., 2005; Chong et al., 2006). In addition to Akt and PKC that have been previously discussed, protein kinase A (PKA) is another potential pathway that may offer control of synaptic plasticity as well as cytoprotection through the mGluR system (Figure
1.13). During paradigms of cellular injury, activation of PKA can prevent apoptosis in a number of cell types, including neurons, neutrophils, and smooth muscle cells (Maiese et al., 1993; Rossi et al., 1995; Orlov et al., 1999). In addition, loss of PKA activity during toxic insults can lead to cell injury (Findik et al., 1995; Nishio & Watanabe, 1997). Protection by PKA is believed to reside upstream from the inhibition of caspase 3-like activity (Parvathenani et al.,
1998). Furthermore, PKA has been shown to phosphorylate Bad, a member of
Bcl-2 protein family, which can prevent the induction of cell injury (Lizcano et al., 2000). Moreover, activation of group-II and group-III mGluR is neuroprotective, whereas group-I mGluR need to be antagonized in order to evoke protection, e.g. using the group-II mGluR agonist LY354740 and mGluR5- selective antagonist MPEP.
25
Figure 1.13 Cytoprotective effects achieved through mGluRs activation. G-protein βγ in the mGluR system activates phospholipase β (PLC-β), diacylglycerol (DAG), and phosphoinositide 3 kinase (PI 3-K). These pathways lead to the activation of protein kinases A (PKA), B (Akt) and C (PKC) that can involve intracellular calcium (Ca2+). mGluRs can activate ERK2 through PKC. PKA can phosphorylate (p) Bad, a member of Bcl-2 protein family, which can prevent apoptotic cell injury. Akt provides an anti-apoptotic survival signal through the phosphorylation and inactivation of Bad and also interfaces with the Wnt pathway. Interconnected pathways with Akt and Wnt involve the forkhead family members (FOXO), mitochondrial (Mito) membrane potential (ΔΨm), cytochrome c (Cyto-c), and caspases. If left unabated, these pathways converge to lead to apoptotic cell injury. (Maiese et al., 2008)
Glial cells predominantly express mGluR1, mGluR3 and mGluR5. In
2+ astrocytes, activation of mGluR5 elicits a rise in [Ca ]i and can induce the release of glutamate and other neurotransmitters, which can evoke synaptic responses in neighbouring cells (Pasti et al., 1997; Bezzi et al., 1998). The in vivo
26 expression of mGluR in oligodendrocytes is unclear. In vitro, OPCs express
GluR1, mGluR2/3, mGluR4 and mGluR5 and they are developmentally downregulated, being high in OPCs and low or absent in immature and mature oligodendrocytes (Deng et al., 2003). One study has reported expression of mGluR1 and mGluR5 in premyelinating oligodendrocytes of postnatal day (P) 6 rat cerebral white matter and in human postmortem preterm tissue (Jantzie et al., 2010). In addition, activation of mGluR5 in an OPC cell line has been shown to evoke Ca2+ oscillations and to be completely lost during differentiation (Luyt et al., 2006). These studies indicated that myelinating oligodendrocytes do not express mGluR, that in OPCs and premyelinating oligodendrocytes are considered to be potentially protective.
1.5 Hypoxia/ischaemia and glutamate excitotoxicity
The brain is amongst the most metabolically active organs in the body, together with heart and liver, despite accounting for only 2% of the body’s mass
(Cunnane et al., 2011). The brain stores little energy in the form of glycogen and relies on circulating glucose and oxygen to meet its metabolic needs (Prins,
2008). When the cerebral blood flow is disrupted (ischaemia) and the oxygen supply is reduced (hypoxia) for prolonged time, neuronal cell death occurs, resulting in a hypoxic brain injury. In adults, stroke is the leading cause of disability and is one of the most frequent causes of death. In neonates, prolonged hypoxia/ischaemia can cause injury resulting in mental retardation, seizures or motor disabilities (Vexler & Ferriero, 2001). One of the most
27 common outcomes is cerebral palsy, which is often caused by periventricular leukomalacia (PVL), an injury of the white matter structures surrounding the ventricles, characterized by early axonal and glial damage (Banker & Larroche,
1962; Volpe, 2001; 2009). Ischaemia can be global or focal. Global ischaemia is the result of a heart arrest and leads to a complete interruption of the cerebral blood flow (CBF), with loss of consciousness. Ten minutes of global ischaemia are fatal for humans; at a cellular level, electrical activity disappears within 40 seconds from the beginning of ischaemia, selective neuronal death can occur and astrocytes become reactive (Rossi et al., 2007; Hertz, 2008). In the case of focal ischaemia, the damage is confined to the area surrounding the occluded vessel. If the focal ischaemia is transient and CBF is restored, the damaged area is reperfused, which constitute a risk in itself because of the production and diffusion of reactive oxygen species (ROS).
In hypoxia/ischaemia, the lack of oxygen and glucose causes a reduction of intracellular adenosine triphosphate (ATP) and cells undergo anoxic depolarization (Rossi et al., 2007). Neurones lose their excitability and all cells lose their capacity to maintain intracellular ion homeostasis (Hansen &
Nedergaard, 1988). This results in a drop in pH, accumulation of extracellular K+, influx of NA+, Ca2+ and Cl- and a massive release of glutamate, result of synaptic release and uptake failure (Hansen & Nedergaard, 1988; Goldberg & Choi, 1993;
Martinez-Sanchez et al., 2004; Bano & Nicotera, 2007). Calcium is pivotal in mediating cell death, whereas glutamate release amplifies the damage by glutamate excitotoxicity. Glutamate activates NMDA and AMPA receptors,
28 which depolarize the cell membrane and activate voltage-operated channels; this leads to further Ca2+ influx, aggravating the intracellular calcium overload.
Increased intracellular Ca2+ promotes the release of additional glutamate, triggering a vicious cycle of glutamate excitotoxicity (Figure 1.14). The cytosolic
Ca2+ is accumulated in the mitochondria, further compromising their ability to produce ATP. Moreover, Ca2+ can stimulate proteolytic enzymes, resulting in fatal damage to the cell membrane, cytoskeleton and DNA.
Figure 1.14 Mechanisms of glutamate excitotoxicity. From Verkhratsky and Butt (2013).
Within the infarcted core, excitotoxicity and necrotic death start after few minutes, whereas in the penumbra, the area surrounding the core, cell death occurs more slowly by apoptosis (Doyle et al., 2008; Hertz, 2008). Like
29 neurones, oligodendrocytes and OPCs are particularly sensitive to ischaemia, most likely because of their inadequate anti-oxidative defences and high levels of AMPA/kainite receptor expression (Fern & Möller, 2000; Back et al., 2001;
Káradóttir et al., 2005; Karadottir et al., 2008). Astrocytes are generally considered to be less susceptible to ischaemia, although there is evidence of astrocytic dysfunction and death following ischaemia, with astrocytes in postnatal ages being particularly vulnerable (Fern, 1998; Zhao & Flavin, 2000;
Bondarenko & Chesler, 2001; Shannon et al., 2007; Salter & Fern, 2008).
1.6 The optic nerve as a model white matter
The focus of this study is CNS white matter, because it is essential for higher brain function and is also disrupted in multiple neuropathologies, including multiple sclerosis, leukodystrophies, stroke, and AD (Bartzokis, 2011b; Butt et al., 2014a). Furthermore, there are numerous reports on the importance of neurotransmitters in white matter pathology, although the precise receptors and mechanisms are largely unresolved (Fern et al., 2014). The optic nerve is one of the most commonly studied white matter preparations and will be used throughout this thesis to study glial characteristics and functions. The optic nerve contains the axons of retinal ganglion cells and the glial cells that support them (Butt et al., 2004, Bolton and Butt, 2005). In tracts such as the optic nerve, most if not all axons are myelinated, whereas other tracts can contain both myelinated and small diameter unmyelinated axons. Developmentally, astrocytes appear first and guide axonal growth to reach their targets. OPCs
30 enter the optic nerve around birth and differentiate into myelinating oligodendrocytes during the first two weeks postnatal (Tsai & Miller, 2002). It is perhaps surprising that neurotransmitter-mediated signalling is prominent in the optic nerve, since there are no neuronal cell bodies or conventional neuron- neuron synapses. However, there is abundant evidence for glutamate- and ATP- mediated neurotransmission in the optic nerve, as well as other neurotransmitters such as GABA and norepinephrine (Figure 1.15).
Figure 1.15 Diversity of neurotransmitters in the optic nerve. From (Butt et al., 2014a).
As described above, on the basis of in vitro studies, there are putative roles for neurotransmission in regulating OPC differentiation and myelination in the optic nerve. In addition, a great deal of our knowledge of the role for
31 glutamate and ATP in white matter pathology comes from the optic nerve (Butt et al., 2014a). However, most studies have been on postnatal optic nerves, and little is known about the precise neurotransmitters expressed in the adult or ageing optic nerve. In addition, the physiological function of neurotransmission in the optic nerve is unresolved, although extensive studies have shown that astrocytes, oligodendrocytes and OPCs make contacts with axons (Butt et al.,
1994a; Butt et al., 1994b; Butt et al., 1999), and that axonal activity activates
2+ glutamate and purine receptors on optic nerve OPCs and can trigger [Ca ]i increase in astrocytes and OPCs (Kriegler & Chiu, 1993; James & Butt, 2001; Butt et al., 2004; Kukley et al., 2007; Ziskin et al., 2007; Hamilton et al., 2008;
Hamilton et al., 2010). The sources of neurotransmitters in white matter and mechanisms of their release remain obscure, but the old dogma that chemical synaptic specializations occur exclusively between neurones has been overturned by studies over the past 20 years. First, there was the discovery that
OPCs form functional glutamatergic and GABAergic synapses with neurones in hippocampus (Bergles et al., 2000; Lin & Bergles, 2004). Later, OPC were found to make occasional synapses with unmyelinated axons in an en passant fashion
(Kukley et al., 2007; Ziskin et al., 2007), and in myelinated axons they contact nodes of Ranvier and respond to axonal activity (Hamilton et al., 2010). To add to the complexity, astrocytes can also have mechanisms of vesicular release of glutamate and ATP (Volterra & Meldolesi, 2005), and optic nerve axons contain glutamate-loaded vesicles and the machinery for vesicular release (Alix et al.,
2008). Combined with evidence that oligodendrocytes and myelin express glutamate receptors, this has led Stys to argue that potential communication
32 between axons and myelin may represent a new type of ‘axon-myelin synapse’ that regulates myelination even in adulthood (Stys, 2011).
1.7 Hypothesis and aims
There is abundant evidence of glutamate-mediated signaling in glial cells and of its importance in white matter neuropathology. However, the majority of studies have been on postnatal tissue and it is unclear whether neurotransmitter signaling mechanisms persists after maturation. Furthermore, despite evidence that glia express mGluR and that they may be therapeutic targets for neuroprotection, their expression and function in white matter glia is unknown. Thus, the hypothesis underpinning this thesis is that mGluR are expressed by white matter glia and may have a protective function against hypoxia/ischemia. The overall aim of the thesis is to test this hypothesis using the mouse optic nerve as a model CNS white matter. Moreover, towards the end of this project, a collaboration with a Spanish research group was established and, as a result, brain tissue from a triple transgenic mouse model of Alzheimer’s disease became available. Therefore, the specific objectives of this study are to:
1. Identify age-related changes in the neurotransmitter profile of the
mouse optic nerve.
2. Examine the expression of functional group I and group II mGluR in
astrocytes and oligodendrocytes in the optic nerve.
33 3. Determine whether mGluR are protective for white matter glia in
hypoxia/ischemia
4. Examine the changes in mGluR5 expression, myelin and OPCs in ageing
and pathology, using a triple transgenic mouse model for Alzheimer’s
disease.
34
Section 2
Materials and methods
35 2.1 Animals
2.1.1 Declaration
Animals were humanely killed by cervical dislocation, in accordance with the regulations issued by the Home Office of the United Kingdom under the
Animals (Scientific Procedures) Act (ASPA), 1986.
2.1.2 Animal Strains
Several different strains of mice were used; C57BL/6J (Wild type black) and transgenic strains;
SOX10-lox-GFP-4xPolyA-lox-DTA, which expresses the enhanced green
fluorescent protein (EGFP) under transcriptional control of the SOX10
gene (Stolt et al., 2006). The SOX10-EGFP transgenic line was a gift from
William D Richardson and Nikoleta Kessaris’ lab (UCL).
TgN (GFAP-EGFP) GFEC-FKi, expressing EGFP under the control of the
GFAP gene promoter, resulting in green astrocytes (Nolte et al., 2001).
TgN (PLP-DsRed1), expressing enhanced red fluorescent protein (DsRed -
drFP583, from Discosoma species) under the control of the PLP gene
promoter, resulting in red myelin (Hirrlinger et al., 2005). The GFAP-
EGFP and PLP-DsRed transgenic lines were a generous gift from Dr.
Frank Kirchhoff (Max Planck Institute for Experimental Medicine,
Gottingen, Germany).
36 NG2-DsRed BAC, in which DsRed is expressed under the control of the
NG2 promoter (Zhu et al., 2008a). The NG2-DsRed transgenic line was a
gift from Dr. Akiko Nishiyama (University of Connecticut, USA).
3xTg AD, which harbours the APP Swedish mutations K670N/M671L, the
presenilin-1 M146V mutation and the tau P301L mutation in the C57BL6
mice background (Oddo et al., 2003) and is a model for Alzheimer’s
disease. 6 months and 24 months 3xTgAD and age-matched brain tissue
was kindly provided by Prof JJ Rodriguez (Ikerbasque, Bilbao, Spain).
2.1.3 Animal Ages
Mice of both sexes but different ages were used throughout this study.
P10-12 (i.e. post natal day 10-12), 1 month or 18 months old mice were used for optic nerve microarray analysis; old optic nerve tissue wasn’t available for the qRT-PCR experiments, therefore the analysis focused on early myelinating tissue
(P7), myelinated tissue (P12) and P30. For the expression studies, P8-9 mice were used for brain/optic nerve sections and optic nerve explants, since the protocol for the latter technique is optimised for pre-myelinated tissue. P7-13 optic nerves were analysed in calcium imaging experiments because of inadequate loading of the calcium sensing dye in older optic nerves. Optic nerves from P8-11 mice were used for the oxygen and glucose deprivation study in order to explore the cytoprotective effect of the activation of mGluR. Finally, in section 6, 6 months and 24 months old 3xTg AD and age-matched control
37 male mice brain tissue was kindly provided by Prof. JJ Rodriguez (Ikerbasque,
Bilbao, Spain), who suggested these two age points as early and late stages of pathology in the triple transgenic mouse model for Alzheimer’s disease.
2.1.4 Tissue Fixation Protocols
Three different fixation protocols have been used in the duration of this project:
a) 1% PFA with 15% Picric acid for 10 mins to fix optic nerve explant cultures
b) 4% PFA with 15% Picric acid to plunge-fix mouse brains (24 hrs) and optic nerves (1 hr) for immunohistochemistry. The fixed tissue was then cryoprotected in 30% sucrose.
c) 4% PFA to plunge-fix optic nerves for 1 hr after oxygen glucose deprivation.
2.2 Molecular Biology
2.2.1 Total RNA Extraction
All tissues, except the P30 optic nerve samples, were disrupted and homogenized using the TissueRuptor and disposable probes of approximately
10 mm diameter (Qiagen, Crawley, West Sussex, UK). Extraction of RNA was performed using the RNeasy Micro Kit (Qiagen) following the manufacturer’s 38 instructions, from approximately 5 mg of mouse cortex, cerebellum and optic nerve. When extracting RNA from P30 optic nerves, the tissue was placed in 500
µL of ice cold Trizol (Sigma) and mechanically disrupted using a sterile plastic pestle (Fisher) and homogenised by being passed through a 23G syringe 10 times on ice. Homogenate was allowed to rest for 5 mins and then passed through a QIAshredder column (Qiagen) for further homogenisation. 100 µL chloroform (Sigma) was added and the homogenate shaken vigorously for 15 secs before being allowed to rest for 3 mins at room temperature. Then, samples were centrifuged at 12000 g for 5 mins at 4°C. Finally, the top clear, aqueous phase containing the RNA was aspirated and transferred into a new 1.5 ml eppendorf where it was mixed at 1:1 ratio with 70% ethanol. The RNeasy
Micro Kit procedure was followed from that point onwards. Approximately 5 mg of tissue was placed in RNA later until it was disrupted and homogenized using the TissueRuptor in a round bottomed collection tube containing a guanidine thiocyanate (GITC) lysis buffer (RLT) and then centrifuged for 3 mins at 13000 rpm. GITC is a chaotropic salt, i.e. a potent protein denaturant, which inactivates RNases thus allowing for the RNA to be isolated intact. The supernatant was carefully removed and placed in a new collection tube before adding 70% molecular grade ethanol at 1:1 ratio. It was then transferred onto an RNeasy spin column containing a silica-gel membrane upon which the RNA would bind during subsequent centrifugation at 10000 rpm for 15 secs. A further wash with 350 µl of a chaotropic salt containing buffer (RW1) was performed at 10000 rpm for 15 secs in order to wash away any remaining contaminating impurities such as degraded proteins and a last was with ethanol
39 was utilised to wash away the salts. A dry spin for 5 mins at full speed was performed to remove all ethanol residues from the membrane. Complete removal of ethanol is essential for high yields of clean eluent. If there is ethanol remaining on the column then the nucleic acids will not be completely rehydrated and elution will result in low yields. Finally, high quality total RNA was eluted in 10µl RNA-free water by centrifuging for 1 min at full speed.
An extra DNase digestion step using DNase I (Qiagen) was performed in order to eliminate genomic DNA that was released in the lysate, as the homogenisation protocol disrupted the nucleic as well as the cell membranes.
All RNA samples were stored at -80°C.
The purity and concentration of isolated total RNA was assessed using a
ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) to measure the UV absorption of samples. The absorption at 260nm (A260) was used to determine the concentration of RNA in undiluted samples. The ratio of absorptions at 260 nm and 280 nm (A260/A280 ratio) was used to determine the samples’ purity, where a ratio of >1.8 indicated high RNA purity. As RNA optimally absorbs light at a wavelength of 260 nm due to its high content of nucleotides, while proteins and other impurities that might be contaminating the isolated RNA absorb best at 280 nm, the A260/A280 ratio is the preferred way to assess if an RNA sample is pure.
40 2.2.2 Quantitative real time PCR (qRT-PCR)
The RT2 First Strand Kit (Qiagen) was used for in vitro transcription of extracted RNA into cDNA to be used for qRT-PCR, following the manufacturer’s instructions. The quantity of RNA that was transcribed was the same for all samples (500 ng). 1350 µl of 2x SYBR Green qPCR Mastermix (Qiagen) was mixed with 102 µL cDNA and 1248 µl ultra-pure water (Ambion) and 25 µL was pipetted in each well of the 96 well-plate arrays. The custom mGluR array used was purchased from SABiosciences (Qiagen) and thermocycled on a Roche
Lightcycler 96 instrument (Roche). The protocol used was the following; 95 °C for 15 secs (1 cycle), followed by 60 °C for 1 min (45 cycles). Data normalisation was performed using the best housekeeping genes for each experiment.
Optimal housekeeping genes were chosen using the NormFinder logarithm
(Andersen et al., 2004) and the standard deviation (SD) method (Mane et al.,
2008). The method of Comparative Quantitation was used to study the expression of metabotropic glutamate receptors with the following formula;
Ct Sample – Ct Calibrator = ∆Ct
Relative quantity = Av(2-∆Ct)
The fold change in mRNA transcript levels between different ages was calculated using the -∆Ct method and tested for significance using ANOVA and post hoc Bonferroni’s tests. Statistical analysis was performed using the Prism 6 software (GraphPad).
41 2.2.3 Microarray
The microarray profiling presented in Section 3 is the result of my analysis on microarrays performed by two of my colleagues, Dr. Virginia
Hawkins and Dr. Andrea Rivera, who collected the postnatal/ageing and adult samples respectively, and carried out the RNA extraction following the Trizol method. Then the samples were sent to the ATLAS-Biolabs company (Germany).
Briefly, 1g of total RNA was used to generate biotinylated cRNA samples, which were hybridized to a GeneChip Mouse Genome 430_2.0 Arrays
(Affymetrix) and visualized using a Affimetryx GeneChip Scanner 3000. This platform contains oligonucleotide probe sets complimentary to the 3’ end of the mRNA transcripts (a 3’-array), with each array containing 45,000 probe sets, corresponding to over 34,000 well-characterized mouse genes. Quality control analysis and data analysis produced .CEL image and >CHP image files for further analysis using an Affymetrix Genechip Operating Software. All samples passed quality control (QC), as reported by Atlas Biolabs (Germany).
Data analysis was performed using the GeneSpring GX 12 Software
(Agilent). Data normalisation was carried out using the MAS-5 algorithm.
GeneSpring GX software was used to profile genes of interest and obtain dataset from samples.
42 2.3 Cell Culture
All cell cultures were completed within a routinely sterilized room containing a laminar flow cabinet (Life Technologies) and cell culture incubator
(Fisher).
2.3.1 Cover Slip Preparation
13mm diameter glass cover-slips were sterilised by immersion in 70% ethanol and placed in 24-well plates (Greiner Bio-One, Manchester, UK) and left to dry in the culture hood. 100 µl of 1% poly L-lysine (Sigma-Aldrich, Dorset, UK) in double distilled sterilised water (ddH2O) was placed on each cover-slip and left overnight at 37°C. Poly L-lysine was removed and replaced with 25 µL of
0.5% laminin (Life Technologies, Paisley, UK) in sterile ddH2O. After two hours, laminin was replaced with 30 µl of sterile ddH2O and the cover-slips were left in the incubator until required. Poly L-lysine coated cover-slips and slides provide higher adhesion, reducing the chance of tissue or cell loss during processing.
This pre-preparation of cover-slips is required when culturing cells that do not adhere well.
2.3.2 Optic Nerve Explant Cultures
Optic nerve explants were cultured as previously described (Greenwood
& Butt, 2003). Briefly, optic nerves were dissected from P8 mice and placed
43 directly into 50µl dissecting medium consisting of high glucose Dulbecco’s
Modified Eagle Medium (DMEM) (Sigma-D5671) containing 10% Fetal Calf
Serum (FCS; Life Technologies), L-Glutamine (Sigma) and 0.1% Gentamycin (Life
Technologies). Next, they were finely chopped with a scalpel blade and triturated with pipettes of decreasing diameter (P1000 then P200) to further break up the tissue. After adding 50 µl more dissecting medium, the solution was pipetted evenly between cover slips (approx. 1 nerve per cover-slip) and left for the initial stick down period. After approximately 24 hrs the dissecting medium was replaced with a low serum (0.5%) modified Bottenstein and Sato
(B&S) culture medium (Bottenstein & Sato, 1979) without thyroid hormones in which 10 ng/ml of recombinant human PDGF-a (R&D Systems) was added to encourage OPC proliferation. After 3-4 days in vitro (DIV) the medium was replaced with B&S medium supplemented with 0.5 mM dbc-AMP (Sigma) until
DIV10.
2.4 Immunostaining techniques
2.4.1 Antibodies
All antibodies used during this project are listed in Table 2.1 and 2.2, along with information concerning host species reactivity, working concentration and source.
44 The secondary antibodies must be raised in the same species as the serum used for blocking, and against the type of animal from which the primary antibody is derived.
Table 2.1 Primary antibodies
Antibody Host Dilution Source Reference
mGluR5 Rabbit 1:1000 Neuromics (Lohith et al., 2013)
mGluR2/3 Rabbit 1:300 Upstate (Meek et al., 2008)
GFAP Chicken 1:500 ChemiconMillipore (Yoshioka et al., 2010) NG2 Rabbit 1:400 Millipore (Gritti et al., 2009)
MBP Rat 1:400 Millipore (Li et al., 2006)
Aβ Mouse 1:1000 Covance (Olabarria et al., 2010)
Table 2.2 Secondary antibodies
Conjugate Reactivity Dilution Source
Gt-a-Rb, Gt-a- AlexaFluor® Life Ck, Gt-a- 1:400 488 Technologies Ms,Gt-a-Rat
Gt-a-Rb, Gt-a- AlexaFluor® Life Ck, Gt-a-Ms, 1:400 568 Technologies Gt-a-Rat
Gt: goat; Rb: rabbit; Ck: chicken; Ms: mouse
45 2.4.2 Immunocytochemistry
Cultured cells were fixed for 10 mins with 1% paraformaldehyde (PFA-
TAAB Laboratories Equipment, UK) dissolved with 15% Picric Acid in 0.2 M
Phosphate Buffer. After washing 3 x10 mins with PBS (Sigma), cells were blocked for 75 mins at room temperature using blocking solution containing 5%
Normal Goat Serum (NGS; Invitrogen) in order to prevent non-specific binding of both the primary and secondary antibodies (Abs) to other antigens on the cells. Where primary antibodies targeted an intracellular epitope of a protein,
0.01% detergent (Triton X, Sigma) was incorporated into the blocking solution, for cell membrane permeabilisation. After blocking, cells were not washed again. Primary antibodies were diluted in PBS. Incubation was performed overnight according to manufacturer’s recommendations at 4°C. Cells were then washed 3 x 10 mins with PBS. The secondary antibody conjugates were diluted in PBS and added to the cells for 1 hr at room temperature on an orbital shaker and protected from light by wrapping the 24-well plate in foil. During this step, the DNA dye Hoechst Blue (Molecular Probes) was added at a concentration of 1:500. After completion of immuno-staining, the cells were once again washed 3 x10 mins with PBS and cover-slips were mounted on clean slides, using mounting medium (Vectashield, Vector Labs, or Fluoromount G,
Southern Biotech).
46 2.4.3 Immunohistochemistry
For the immunohistochemical analysis presented in Section 4, mouse brains and optic nerves were dissected and fixed with submersion in 4% PFA with 15% picric acid for 24 hrs and 1 hr respectively. After fixation, tissues were washed 3 times in PBS and stored in 30% sucrose in PBS at 4°C for 24-48 hrs.
Brains and optic nerves were embedded in cryo-m-bed on a cork board and stored at -80C until required for use. Coronal brain sections and longitudinal optic nerve sections (14μm) were cut using a Leica cryostat and collected onto poly-l-lysine coated slides. Samples were stored at -80C until assayed. Tissue was blocked with 10% serum (NGS or NDS) for 1-2 hrs, washed 3 times in PBS, and incubated overnight in the antibody solution, which comprised of the primary antibody diluted in blocking solution containing Triton-X. Tissues were then washed 3 times again in PBS and incubated with secondary antibody diluted in blocking solution for 1 hr at room temperature on an orbital shaker and protected from the light. Following secondary antibody incubation, tissues were washed 3 times with PBS before being covered with mounting medium and glass coverslips ready for imaging.
The 3xTgAD and age-matched control brains were kindly provided by
Prof. JJ Rodriguez (Ikerbasque, Bilbao, Spain) and required another protocol for immunohistochemistry, due to a different method of perfusion, as described in
(Noristani et al., 2012). Male 3xTgAD and non-transgenic control mice were anaesthetized with intra-peritoneal injection of sodium pentobarbital (50 mg kg) at 6 and 24 months of age. The mice were perfused through the aortic arch
47 with 3.75% acrolein (TAAB, Berkshire, UK) in a solution of 2% paraformaldehyde
(Sigma, Cambridge, UK) and 0.1 M phosphate buffer (PB) pH 7.4, followed by 2% paraformaldehyde. Brains were then removed and cut into 4- to 5-mm coronal slabs of tissue containing the entire rostrocaudal extent of the hippocampus.
The brain sections were postfixed in 2% paraformaldehyde for 24 h and kept in
0.1 M PB, pH 7.4. Coronal sections of the brain were cut into 40 m thickness using a vibrating microtome (VT1000S; Leica, Milton Keynes, UK). Free floating brain sections in 0.1 M PB, pH 7.4, were collected and stored in cryoprotectant solution containing 25% sucrose and 3.5% glycerol in 0.05 M PB at pH 7.4.
Coronal sections at levels -1.58 ⁄ -2.46 mm (hippocampus) posterior to bregma were selected for immunohistochemistry according to the mouse brain atlas of
Paxinos & Franklin. The sections were incubated for 30 min in 30% methanol in
0.1 M PB and 3% hydrogen peroxide (H2O2) (Sigma). Sections were then rinsed with 0.1 M PB for 5 min and placed in 1% sodium borohydride (Sigma) for 30 min. The sections were then washed with PB profusely before rinsing in 0.1 M
Trizma base saline (TS) for 10 min. Brain sections were then incubated in 0.5% bovine serum albumin (BSA) (Sigma) in 0.1 M TS and 0.25% Triton (Sigma) for 30 min. Sections were incubated for 24 h at room temperature in primary antibody. Tissues were then washed 3 times again in TS and incubated with secondary antibody diluted in blocking solution for 1 hr at room temperature on an orbital shaker and protected from the light. Following secondary antibody incubation, tissues were washed 2 times with TS and 3 times with PB before being covered with mounting medium and glass coverslips ready for imaging.
48 2.4.4 Confocal Microscopy
Fluorescence-stained cells and sections were examined under a confocal microscope with the following characteristics (Table 2.3);
Table 2.3 Confocal microscope characteristics
Zeiss Axiovert LSM 710 VIS405, Laser confocal system Microscope and LED Light Source Zeiss LSM 5 Pascal (Colibri) Axioskop 2
Argon, helium-neon Sequential excitation at Lasers (HeNe1) and diode wavelengths 488nm, lasers 568nm and 405nm
x20 (air), or x40, x63 and Zeiss LSM 510NLO Image Acquisition x100 (oil immersed) confocal scan head objectives
LSM and Zen 9000 Image analysis Zeiss Software Software
2.4.5 Immunohistochemistry negative controls
Immunohistochemistry is a powerful investigative tool that can provide supplemental information to the routine morphological assessment of tissues.
The application of antibodies requires adaptation and refinement of immunohistochemical protocols, particularly for use in fixed tissues. In contrast
49 to solution-based immunoassays that detect relatively abundant native proteins, in fixed tissues the preservation of antigen is variable and unpredictable. In order to avoid non-specific staining, well-characterised antibodies were used throughout this study (see references in Table 2.1).
Moreover, a negative control, omitting the primary antibody, was performed in every essay (Figure 2.1).
50
Figure 2.1 Negative controls for immunohistochemistry. Representative images of negative controls, in which primary antibodies were omitted. Panels A and B show negative controls for mGluR2/3 and mGluR5 respectively, on optic nerve sections from a P8 GFAP-EGFP reporter mouse. Negative controls for GFAP, NG2 and MBP were carried out on brain slices from 6 months old non transgenic mouse (C, D and E), whilst the control for the A antibody was performed on 24m 3xTgAD brain tissue. Scale bars: 50 m. 51 2.4.6 Morphological analysis of OPCs
The volume and the surface of individual NG2 labeled OPCs was estimated on 3-dimentional reconstructed images obtained with confocal scanning microscopy (Zeiss 710), recording optical sections at 0.2 µm steps.
Parallel confocal planes were superimposed and morphological analysis was carried out by Cell analyst software (Chvátal et al., 2007). Digital filters (average
3x3, convolution, gauss 5x5, despeckle, simple objects removal) were used to determine surface and volume of the NG2-stained oligodendrocyte progenitor cells, as well as the surface and the volume of their soma.
2.4.7 Statistical analysis
Data are expressed as mean ± SEM. The Gaussian distribution of the data was analysed with the D’Agostino & Pearson’s normality test. Unpaired t- test analysis was used to examine the differences in the surface area and volume of NG2 labeled cells between the 3xTg-AD and non-transgenic animals.
Difference between the groups were considered as significant if p≤0.05. The statistical analysis was performed using Prism 6.0 Software (GraphPad Software
Inc.).
52 2.5 Calcium Imaging
2.5.1 Optic nerve dissection and dye loading
Optic nerves were excised from P7-P13 wild type C57BL/6 mice and incubated for 1 hr in aCSF containing 4 µM Fluo-4 AM (Molecular Probes) which is a green-fluorescent Ca2+ dye in its acetoxymethyl ester form. In this form, an acetoxymethyl ester group renders the dye membrane permeable. Once taken up by a cell, cytosolic esterases remove this ester group and the dye remains intracellular (Garaschuk et al., 2006).
2.5.2 Imaging parameters
Dye loaded nerves were washed in aCSF to remove excess dye, and then placed in a perfusion chamber under a Zeiss LSM 5 Pascal Axioskop 2 confocal microscope and continuous perfusion with aCSF in the bath was established. A
20x/0.50 WPh2 Achroplan water immersion lens objective was used to visualise the intracellular Ca2+ changes after excitation of the optic nerves at 488 nm for
Fluo4. Optical z-sections (typically 7-8 sections at 2–3 µm intervals) were obtained using the Zeiss LSM Image Examiner software (Zeiss, Germany).
Regions of interest (ROI) covering single-cell bodies were selected within the z- stacks and the change in fluorescence intensity was measured.
53 2.5.3 Typical experiment
The nerve was continuously bathed in aCSF via a multitap system that allowed rapid turnover to solutions containing agents of interest (listed in Table
2.4). The health of the optic nerves was assessed by briefly (30 secs) inducing an
ATP response at the beginning and end of each experiment and in the cases that
2+ [Ca ]i increase was not observed in response to ATP, the nerve was excluded from analysis. In any given cell, the response to ATP provided a measure of the
2+ maximal rise of glial [Ca ]i, irrespective of the level of Fluo4 loading (Hamilton
2+ et al., 2008). Thus, quantification of the change in glial [Ca ]i could be measured as the change in fluorescence from baseline (∆F/F), and data expressed relative to the ATP response in the same cell, so that comparison can be made between treatments.
2.5.4 Solutions
aCSF (in mM): NaCl,133; KCl, 3; CaCl2, 1.5; NaH2PO4, 1.2; MgCl2, 1.0;
D-glucose. 10; HEPES, 10; pH 7.3 NaOH.
See Table 2.4 for agonists and antagonists.
54 Table 2.4 List of pharmacological agents used in calcium imaging
Name Concentration used Function Company
ATP 100µM P2 purinoceptor Sigma Adenosine 5'-triphosphate agonist. disodium salt ACPD Selective agonist for both group I and 100µM Tocris (±)-1-Aminocyclopentane- group II mGlu trans-1,3-dicarboxylic acid receptors
DHPG Selective group I metabotropic 100µM Tocris (RS)-3,5- glutamate receptor Dihydroxyphenylglycine agonist
AIDA A relatively potent and selective (RS)-1-Aminoindan-1,5- 100µM antagonist of group I Tocris dicarboxylic acid metabotropic glutamate receptors
2.5.5 Statistical analysis
Measurements were made from multiple cells (n>15 ROIs) in each nerve and experiments were replicated in n4. Data were then expressed as mean
SEM, where n represents the number of cells, and tested for significance using the paired t-test (Graphpad 6.0 software, Prism).
2.6 Oxygen and Glucose Deprivation (OGD)
Optic nerves from P8-11 wild type C57BL/6 and transgenic GFAP-EGFP,
Sox10-EGFP, PLP1-dsRed mice were isolated intact and immediately placed in aCSF for 30 mins at 37°C in a normoxic incubator (95% air/5% CO2) and allowed to stabilise. Control groups were incubated for a further 1 hr in normal aCSF containing glucose with 5% CO2/95% O2 (oxygenated, O2). Oxygen-glucose 55 deprivation (OGD) was achieved using the method of Fern and colleagues
(Salter & Fern, 2005), by incubating nerves for 1 hr at 37 °C in glucose-free aCSF
(osmolarity was maintained by replacing glucose with sucrose), and switching the chamber atmosphere to 5% CO2/95% N2.
Pharmacological modulators added to the aCSF solution during O2 and
OGD incubations are listed in Table 2.4.
56 Table 2.5 List of pharmacological agonists and antagonists used in OGD
Name Concentration used Function Company
ACPD Selective agonist for 100µM both group I and group II Tocris (±)-1-Aminocyclopentane-trans- 1,3-dicarboxylic acid mGlu receptors DHPG Selective group I 100µM metabotropic glutamate Tocris (RS)-3,5-Dihydroxyphenylglycine receptor agonist LY 379268 100µM Highly selective group II Tocris (1R,4R,5S,6R)-4-Amino-2- mGlu receptor agonist oxabicyclo[3.1.0]hexane-4,6- dicarboxylic acid Go6976 Potent protein kinase C 5,6,7,13-Tetrahydro-13-methyl- 10µM (PKC) inhibitor; Tocris 5-oxo-12H-indolo[2,3- selectively inhibits a]pyrrolo[3,4-c]carbazole-12- PKCα and PKCβ1 propanenitrile MRS2365
[[(1R,2R,3S,4R,5S)-4-[6-Amino-2- 100µM Highly potent, selective Tocris (methylthio)-9H-purin-9-yl]-2,3- dihydroxybicyclo[3.1.0]hex-1- P2Y1 receptor agonist yl]methyl] diphosphoric acid mono ester trisodium salt MRS2279
(1R*,2S*)-4-[2-Chloro-6- Selective, high affinity (methylamino)-9H-purin-9-yl]-2- 100µM competitive antagonist Tocris (phosphonooxy)bicyclo[3.1.0]hex ane-1-methanol dihydrogen of the P2Y1 receptor phosphate ester diammonium salt dbcAMP Cell-permeable cAMP 100 µM analog that activates Sigma N6,2′-O-Dibutyryladenosine 3′,5′- cyclic monophosphate sodium cAMP dependent salt protein kinase (PKA). Forskolin
[3R- (3α,4aβ,5β,6β,6aα,10α,10aβ,10b 100µM Cell-permeable activator Sigma α)]-5-(Acetyloxy)-3- of adenylyl cyclase ethenyldodecahydro-6,10,10b- trihydroxy-3,4a,7,7,10a- pentamethyl-1H-naphtho[2,1- b]pyran-1-one
57 2.6.1 Quantification of glia
At the end of the 60 mins incubation period, optic nerves were fixed and whole-mounted for image capture using a Zeiss LSM 710 metaconfocal microscope (as previously described). Cells expressing the GFAP-EGFP and
SOX10-EGFP or PLP1-dsRed reporters were visualised at 488 nm or 568 nm, respectively, using an argon laser and x40 oil immersion lens with high numerical aperture (1.3 nm); images were captured maintaining the acquisition parameters constant between samples. In each nerve, the total number of cells was counted in two FOV along the length of the optic nerve. The FOV comprised a constant volume of 20 x 20 µm in the x-y-plane and 15 x 1 µm optical sections in the z-plane, in the centre of the optic nerve. Cell counts are expressed as mean number of cells per FOV + SEM (n>5, where ‘n’ represents the number of nerves). The Gaussian distribution of the data was analysed with the D’agostino
& Pearson’s normality test and the Bartlett’s test was performed to investigate whether all the populations had the same standard deviation. The significance was determined using the parametric One-way ANOVA and, in order to compare more than three samples, the Newman–Keuls multiple comparison post-hoc analysis was performed, (as published in (Hawkins & Butt, 2013), using
Graphpad Prism 6.0.
58
Section 3
Microarray Analysis of Age-Related
Changes in Neurotransmitter Signalling
Mechanisms in the Mouse Optic Nerve
59 3.1 Introduction and aims
CNS white matter comprises tracts of myelinated axons that interconnect neurones from one area of the brain to another. One of the most commonly studied white matter preparations is the optic nerve, which connects the retina to the brain and contains the axons of retinal ganglion cells (RGC) that synapse in the superior colliculus in the mouse and lateral geniculate nucleus in human. The vast majority of optic nerve axons are myelinated in the adult, and the main cells associated with them are the myelinating oligodendrocytes and astrocytes, which make up over 90% of the total cells, together with small populations of OPCs (~5%) and microglia (Butt et al., 2004). Astrocytes appear first in development, followed by OPCs that differentiate into myelinating oligodendrocytes mainly during the first two weeks postnatal, although they continue to generate oligodendrocytes slowly throughout adult life (Butt &
Ransom, 1993; Rivers et al., 2008). Notably, the optic nerve does not contain neuronal cell bodies or synapses, although this is not the case for all white matter structures (von Engelhardt et al., 2011). It is surprising, therefore, that neurotransmitter-mediated signalling is prominent in the optic nerve and other white matter areas studied, since neurotransmitter signalling is generally considered to be an exclusive function of neurones confined to synapses (Butt et al., 2014b).
The primary physiological functions of neurotransmitters in white matter remain elusive, although there are reported roles for glutamate, ATP,
60 and GABA in regulating oligodendrocyte differentiation and myelination (Butt,
2006; 2011; Butt et al., 2014b). Moreover, clear roles for ATP and glutamate have been demonstrated in oligodendrocyte pathology, most notably in multiple sclerosis (MS) and hypoxia/ischaemia (Matute, 2011). However, the majority of studies on white matter are during development and details of neurotransmitter signalling mechanisms in adult and ageing white matter are lacking. Studies in primates and rodents provide indirect evidence of myelin loss in the ageing brain, which may be due to deregulation of oligodendrocyte generation from OPCs (Peters & Sethares, 2004; Rivers et al., 2008). Notably,
MRI and post-mortem human studies have identified myelin loss as a key feature of the normal ageing brain and there is evidence this is accelerated in dementia (Ihara et al.; Bartzokis et al., 2004; Bowley et al., 2010). These studies raise the possibility that neurotransmitter signalling, which is proposed to regulate oligodendrocyte differentiation and integrity, may be deregulated in ageing white matter.
The aim of this section is to use Affymetrix GeneChip arrays to profile genes involved in neurotransmitter signaling during development and in the ageing mouse optic nerve.
61 3.2 Results
3.2.1 Microarray analysis of gene expression in mouse optic nerve
To better understand the potential age-related changes in neurotransmitter signaling in white matter, a three-way Affymetrix GeneChip analysis was used to profile mRNA isolated from optic nerves from mice aged
10-12 days (P10-12, postnatal), 1-month (adult) and 18-month (ageing).
Following the protocol of Barres and colleagues (Zamanian et al., 2012), total
RNA was extracted from acutely dissected optic nerves pooled from n=6-12 animals for each sample. Total RNA concentration was determined by spectrophotometry and samples were sent to Atlas Biolabs (Germany) for microarray analysis. Briefly, 1g of total RNA was used to generate biotinylated cRNA samples, which were hybridized to a GeneChip Mouse Genome 430_2.0
Arrays (Affymetrix) and visualized using a Affimetryx GeneChip Scanner 3000.
This platform contains oligonucleotide probe sets complimentary to the 3’ end of the mRNA transcripts (a 3’-array), with each array containing 45,000 probe sets, corresponding to over 34,000 well-characterized mouse genes. All samples passed quality control (QC), as reported by Atlas Biolabs (Germany). For 1- month tissue, two samples were available and replicates were performed, but this was not feasible for P10-12 and 18-month tissue, due to the availability of tissue. The generated CEL file was analysed with Genespring GX 12 (Agilent) software, using the MAS-5 algorithm to generate expression values and absent/present (A/P) calls (Hubbell et al., 2002). Expression profiles were then
62 analysed using fold change (FC), A/P calls and profile plots, all within Genespring
GX 12 software (Agilent).
Data analysis using the MAS-5 algorithm showed expression values for
45,101 entities (probe sets) per sample, of which at least 28,302 were present in one or more of the samples. Expression values (E, Absolute value) are summarized by baseline transformation to the median of all samples as demonstrated in Figure 3.1, with the box and whisker plot showing the spread of the values (Figure 3.1A) and the Venn diagram illustrating the number of A/P entities common and exclusive to the three age groups (Figure 3.1B). Lists of all entities present or absent in each group were then generated for further analysis. For example: 24,369 entities present in all samples; only 707 entities were specific to ageing nerves, whilst 3455 were specific to adult nerves and
1345 to postnatal nerves; 4136 entities were common to adult and ageing nerves, but absent in postnatal nerves, whilst only 179 entities were common to both postnatal and ageing nerves and absent in adult nerves. The profile plot illustrates these changes in entity expression between the samples, with all entities illustrated in Figure 3.1C, and 20,736 entities identified by filtering on fold-change (FC), with a cut-off value of 1.5 FC (Figure 3.1D).
63
Figure 3.1 Gene expression analysis of P10-12, 1-month and 18-month mouse optic nerve. Pooled optic nerves from 6-12 animals per group were assessed, using the genome wide Mouse Affymetrix GeneChip microarray and scanned intensity values were normalized using the MAS-5 algorithm. (A) Box-whisker plot of the data range of all probes in P10-12, 1-month and 18-month samples. (B) Venn diagram of the probes catalogued by their presence in each group. (C, D) Profile plots of changes in probe intensity values between the samples, with all probes (C) and probes with 1.5 fold- change (FC) across the groups (D).
64 3.2.2 Macroglial genes expression in optic nerve samples
The bulk of RNA extracted from optic nerves is from astrocytes and oligodendrocytes, which represent >90% of the cells, with low levels of RNA from OPC, microglial and vascular cells and axons (Salter & Fern, 2005). To confirm this, the expression levels of well-described glial markers are compared with neurone/axon and endothelial markers in postnatal, adult and ageing optic nerves (Figure 3.2) and the P/A calls given in Table 3.1. In all the groups, the probes with the highest level of expression were for astrocyte- and oligodendrocyte-specific genes, while the expression of non-glial markers was marginal. Neuronal and axonal markers were mainly called absent or were present at negligible levels (E<50), although the presence of the axoskeletal
Ank3 (Ankyrin3G) and Nefl (Neurofilament, light chain) are consistent with axonal mRNA being present in the optic nerve (Salter & Fern, 2005), and these were particularly prominent in the postnatal nerve. Furthermore, although the nodal axonal sodium and potassium channels Kcnq2 (Kv7.2) and Scn8a (Nav1.6) were called absent at all ages, the synaptic vesicle marker Sv2b was prominently expressed in the postnatal nerve, consistent with evidence of axonal vesicular release mechanisms described in the developing optic nerve (Alix et al., 2008).
Similarly, the endothelial marker Cldn5 (Claudin5), an important component of endothelial tight junctions in the blood-brain barrier (BBB), was highly expressed in the postnatal nerve; indeed, all endothelial markers were more highly expressed in the postnatal nerve, consistent with increased vascularization during the major period of nerve growth. Notably, in the
65 postnatal nerve the levels of expression of Cldn5 and Ank3 are similar to the levels of Kcnj10 (Kir4.1), the main astrocyte potassium channel, indicating axonal and vascular transcripts are major components of the postnatal nerve.
Interestingly, the microglial markers tested were not expressed at high levels, with the notable exceptions of Mrc1 (CD206), which was highly expressed in the postnatal nerve and downregulated thereafter, and Fcgr3 (CD16), which was low in postnatal and adult nerves, but markedly upregulated in ageing nerves.
These results suggest an age-related shift in the microglial function, although this did not appear to be a clear-cut shift between the M1 (activated, or pro- inflammatory) and M2 (alternatively activated or anti-inflammatory, involved in restoring tissue homeostasis) phenotypes; Mrc1 and Fcgr3 are both phagocytic markers, whilst Arg1 and iNOS, important markers of M2 and M1 microglial activation, respectively, were called absent, and the M1 markers Cd86 and
Fcgr2b (CD32) were expressed at varying levels in the different age group (Kigerl et al., 2009).
In addition to evident age-related changes in the expression of axonal, endothelial and microglial markers, expression levels of the main astrocyte, oligodendrocyte and OPC markers were also at their highest in the postnatal nerve (Figure 3.2; Table 3.1). Further analysis of the overall proportion of entities in the different cell types indicates an overall trend in the reduction of oligodendrocyte genes with age, together with proportional increases in astrocyte genes between the postnatal and adult nerves, and in microglial genes in ageing nerve (Figure 3.3). The myelin- and OPC-related genes were
66 highest in the postnatal optic nerve, during the period of maximum active myelination, decreasing in adulthood and then diminishing further in the ageing nerves; a notable feature was the downregulation of OPC genes Pdgfra and
Nkx2.2 in the ageing nerve, and that Cspg4 (NG2) was called absent in all samples.
67
Figure 3.2 Gene expression analysis of well described cell specific marker in P10-12, 1-month and 18-month mouse optic nerve. Pooled optic nerve transcript from 6-12 mice was assessed using a genome wide microarray and scanned intensity values were normalized using the MAS-5 algorithm. See Table 3.1 for protein names.
68
Table 3.1 Gene expression levels of well described cell markers for astrocytes, oligodendrocytes, OPCs, neurons, microglia and endothelial cells.
Gene Symbol P10-12 1mo 18mo Name Cell type E Call E Call E Call Kcnj10 1505.75 P 1014.27 P 1084.17 P Kir4.1 Astrocyte Aqp4 2154.99 P 594.41 P 584.36 P Aquaporin 4 Astrocyte Gfap 7788.83 P 1798.69 P 2110.47 P GFAP Astrocyte Gja1 2740.18 P 1551.12 P 1349.98 P Cx43 Astrocyte Slc1a3 2112.81 P 395.78 P 431.36 P GLAST/EAAT1 Astrocyte Cnp 9261.22 P 1645.40 P 1652.92 P Cnpase Oligodendrocyte Mag 7159.49 P 940.08 P 583.02 P MAG Oligodendrocyte Plp1 8534.50 P 2243.87 P 2274.25 P PLP Oligodendrocyte Mbp 7316.75 P 1694.00 P 2013.73 P MBP Oligodendrocyte Mobp 7069.88 P 1606.95 P 1674.88 P MOPBP Oligodendrocyte Mog 4026.13 P 2010.19 P 1495.71 P MOG Oligodendrocyte Ugt8a 5394.73 P 936.74 P 780.83 P UDP glycosyltransferase Oligodendrocyte Cspg4 233.98 P 37.85 A 34.00 A NG2 OPC Pdgfra 657.60 P 221.11 P 80.95 P PDGFRa OPC Nkx2-2 1259.47 P 196.29 P 86.28 P Transcription factor OPC Sox10 1317.37 P 466.09 P 444.52 P Transcription factor OPC/OL Olig2 1207.22 P 299.41 P 125.87 P Transcription factor OPC/OL Kcnq2 15.97 A 2.94 A 23.48 A Kv7.2, nodal K+ channel Axon Scn8a 44.75 A 18.53 A 33.47 A Nav1.6, nodal Na+ channel Axon Cntnap1 9.30 A 4.22 A 7.01 A Caspr/Contactin Axon Ank3 989.48 P 275.20 P 139.77 P Ankyrin 3G, nodal protein Axon Nefl 466.09 P 152.02 P 364.95 P Neurofilament, light chain Axon Slc12a5 51.11 P 7.99 A 130.78 P K+/Cl- transporter Neuron Sv2b 111.52 P 35.68 A 71.83 P Synaptic vesicles Neuron Syt1 55.81 P 4.67 A 132.26 P Synaptotagmin 1 Neuron Mrc1 1036.07 P 116.24 P 96.56 P CD206 Microglia/M2 Arg1 5.11 A 4.46 A 8.10 A Arginase 1 Microglia/M2 Fcgr3 309.30 P 328.40 P 1079.64 P CD16 Microglia/M2 Nos2 2.75 A 1.00 A 1.00 A iNOS Microglia/M1 Cd86 66.06 P 26.60 A 121.34 P CD86 Microglia/M1 Fcgr2b 196.23 P 93.58 P 175.11 P CD32 Microglia/M1 Cldn5 1638.45 P 204.13 P 205.19 P Claudin 5 (tight junctions) Endothelium Ocln 204.02 P 50.12 A 14.16 A Occludin (tight junction) Endothelium Pecam1 165.14 P 8.28 P 6.02 A adhesion molecule Endothelium Tek 231.34 P 116.33 P 47.17 A tyrosine kinase Endothelium Vwf 455.64 P 239.24 P 164.16 P Von Willebrandt factor Endothelium
69
Figure 3.3 Age-related changes in the proportional expression of markers for astrocytes, oligodendrocytes, OPCs, axons, microglia and endothelial cells in mouse optic nerve. The expression values of the top five markers were summed for each cell type and expressed as a percentage.
70 3.2.3 Most expressed genes by glial cells throughout aging
Microarray data were analysed to obtain the top 100 genes that are most expressed in young, adult and aging optic nerves. Figure 3.4 shows how some of the most expressed entities are exclusive to a particular age group, suggesting a specific functional role of their coding protein at that age, whilst others are highly expressed in two or all three age groups. Gene symbols, expression levels and coding protein role for the top 100 probes expressed in P10-12, 1-month and
18-month optic nerves are listed respectively in Tables 3.2, 3.3 and 3.4. The genes were classified in categories according to their coding protein biological function, summed and then expressed as percentage in Figure 3.5. This analysis shows that the majority (35-63%) of the top 100 expressed genes at all three ages are involved in gene expression itself, having a role in transcription, translation and protein folding. In all three groups, many of the most expressed genes have a role in the primary glial functions of myelination, control of cellular homeostasis and metabolism, cytoskeleton and intermediate filament formation and protein degradation. The proportion of genes involved in myelination and protein degradation is at its highest at P10-12 (15% and 4%) and decreases markedly in ageing optic nerves (5% and 1%), consistent with a transition from an actively developing and remodelling environment in the postnatal nerve to a less plastic and more stable mature tissue.
71
Figure 3.4 Venn diagram illustrating the distribution of the top 100 most expressed entities in P10-12, 1-month and 18-month optic nerve.
72 Table 3.2 Expression levels of the top 100 genes in P10-12 optic nerves.
Probe Set ID Gene Symbol E Coding protein role/function
1456228_x_at Mbp 12737.34 myelination 1416589_at Sparc 11961.59 ossification 1450641_at Vim 11353.2 intermediate filament 1424635_at Eef1a1 11042.74 translational elongation/protein homooligomerization 1433785_at Mobp 10785.7 intracellular protein transport/nervous system development 1437341_x_at Cnp 10768.35 myelin associated enzyme 1433472_x_at Rpl38 10659.44 regulation of translation 90S preribosome assembly 1416642_a_at Tpt1 10366.71 cell survival 1449436_s_at Ubb 10302.05 protein degradation 1433443_a_at Hmgcs1 10271.65 ketogenesis/metabolism 1422156_a_at Rps2 10235.47 ribosomal small subunit assembly/formation of translation preinitiation complex/translation 1415906_at Tmsb4x 10137.1 regulation of actin polymerization 1437413_x_at Rps29 10113.04 translation 1460008_x_at Rpl31-ps12 9990.166 translation/ protein folding 1428792_at Bcas1 9868.191 breast carcinoma-amplified sequence 1 1436722_a_at Actb 9831.437 actin formation 1423860_at Ptgds 9805.28 prostaglandin biosynthetic process 1417184_s_at Hbb-bs/Hbb-bt 9756.646 hemoglobin 1436924_x_at Rpl31-ps12 9698.184 translation/protein folding 1451961_a_at Mbp 9680.358 myelination 1425467_a_at Plp1 9643.99 axon ensheathment/myelination 1432827_x_at Ubc 9643.519 protein degradation 1416624_a_at Kxd1/Uba52 9621.252 translation 1418884_x_at Tuba1a 9507.544 microtubule formation 1417451_a_at Ppia 9471.375 protein peptidyl-prolyl isomerization/protein folding 1438859_x_at Rps29 9437.384 translation 1431765_a_at Rps2 9436.734 ribosomal small subunit assembly/formation of translation preinitiation complex/translation 1415833_x_at Rps29 9400.983 translation 1426508_at Gfap 9327.452 intermediate filament 1422475_a_at Rps3a1 9264.441 translation 1451718_at Plp1 9257.401 axon ensheathmen/myelination 1415879_a_at Rplp2 9206.688 translational elongation 1449296_a_at Cnp 9188.181 microtubule cytoskeleton organization/axonogenesis 1455578_x_at Rpl41 9136.645 translation 1437185_s_at Tmsb10 9128.296 cytoskeleton organization 1415822_at Scd2 9125.701 lipid metabolic process/fatty acid metabolic process/myelination 1450857_a_at Col1a2 9073.227 collagen formation 1415701_x_at Rpl23 9054.395 translation 1460175_at Rps23 9021.685 translation 1422613_a_at Rpl7a 9002.883 ribosome biogenesis 1428361_x_at Hba-a1/Hba-a2 8986.983 hemoglobin 1436995_a_at Rpl26 8951.73 rRNA processing/translation 1455976_x_at Dbi 8920.191 triglyceride metabolic process 1454859_a_at Rpl23 8907.353 translation 1418625_s_at Gapdh 8882.264 glucose metabolic process/glycolysis 1433532_a_at Mbp 8866.951 myelination 1459986_a_at Rps17 8857.918 ribosomal small subunit assembly/translation 1416277_a_at Rplp1 8833.914 translational elongation 1416141_a_at Rps6-ps4 8823.625 G1/S transition of mitotic cell cycle 1418000_a_at Itm2b 8790.517 apoptotic process/negative regulation of amyloid precursor protein biosynthetic process 1419646_a_at Mbp 8754.418 myelination 1415812_at Gsn 8695.245 actin assembly and disassembly 1426088_at ND5 8688.919 electron transport chain 1416420_a_at Rpl9 8639.12 translation 1454651_x_at Mbp 8635.563 myelination 1417275_at Mal 8632.116 induction of apoptosis/myelination 1451101_a_at Rps28 8631.813 rRNA export from nucleus/translation 1416330_at Cd81 8628.887 activation of MAPK activity 1438649_x_at Pebp1 8624.359 MAPK cascade/regulation of neurotransmitter levels/negative regulation of protein phosphorylation 1448771_a_at Fth1 8611.063 iron ion transport and homeostasis 1436263_at Mobp 8570.687 intracellular protein transport/nervous system development 1436201_x_at Mbp 8496.671 myelination 1423763_x_at Rps28 8489.383 rRNA export from nucleus/translation 1423507_a_at Sirt2 8421.868 protein deacetylation 1453723_x_at Ubb 8412.609 protein degradation 1450840_a_at Rpl39 8392.408 translation 1450088_a_at Mobp 8392.092 intracellular protein transport/nervous system development 1416404_s_at Rps16-ps2 8386.945 rRNA processing/translation 1455789_x_at Hspa8 8371.99 cell morphogenesis/ATP catabolic process/transcription, DNA-dependent 1436064_x_at Rps24 8367.11 maturation of SSU-rRNA from tricistronic rRNA transcript/rRNA processing 1455662_x_at Rps17 8365.075 ribosomal small subunit assembly/translation 1415942_at Rpl10 8361.41 translation 1423846_x_at Tuba1b 8352.836 microtubule formation 1448152_at Igf2 8313.95 development and growth
73 1449323_a_at Rpl3 8307.013 translation/cellular response to interleukin-4 1435151_a_at Rps3 8296.515 translation/induction of apoptosis/response to DNA damage stimulus 1425742_a_at Tsc22d1 8286.702 TGF beta 1 induced trascriptional repressor 1454778_x_at Rps28 8283.73 rRNA export from nucleus/translation 1455997_a_at Uqcrb 8283.022 mitochondrial electron transport 1455485_x_at Rpl13a 8280.98 transcription, DNA-dependent/regulation of translation 1437005_a_at Rpl18 8278.179 translation/oxidation-reduction process 1455106_a_at Ckb 8251.615 energy homeostasis 1435643_x_at Ubb 8231.305 protein degradation 1416099_at Rpl27 8224.691 translation 1432466_a_at Apoe 8209.396 lipid metabolism 1456142_x_at Morf4l1 8208.23 chromatin remodeling/transcriptional activation 1449196_a_at Rps27a 8187.117 translation/ubiquitin homeostasis 1433721_x_at Rps21 8145.562 translation/ribosomal small subunit biogenesis 1423665_a_at Rpl5 8123.343 rRNA processing/translation 1437706_x_at Rps14 8119.005 ribosomal small subunit assembly/regulation of translation 1434396_a_at Myl6 8105.839 regulatory light chain of myosin 1428161_a_at Chchd2 8096.821 Coiled-Coil-Helix-Coiled-Coil-Helix Domain-Containing Protein 2 1416074_a_at Rpl28 8093.007 translation 1428212_x_at Rpl31-ps12 8038.415 translation 1448739_x_at Rps18 8025.492 translation/ribosome biogenesis 1420830_x_at Ywhaq 8007.406 protein targeting/small GTPase mediated signal transduction 1416219_at Rpl19 7980.535 translation 1419441_at Rplp0 7979.316 translational elongation/ribosome biogenesis/cellular response to interleukin-4 1454661_at Atp5g3 7974.935 ATP synthesis coupled proton transport 1423221_at Tubb4a 7969.454 microtubule formation
74 Table 3.3 Expression levels of the top 100 genes in 1-month optic nerves.
Probe Set ID Gene Symbol E Coding protein role/function
1433532_a_at Mbp 2989.54 myelination 1433785_at Mobp 2947.40 intracellular protein transport/nervous system development 1448771_a_at Fth1 2939.68 iron ion transport and homeostasis 1455976_x_at Dbi 2647.00 triglyceride metabolic process 1433432_x_at Rps12 2626.77 translation 1438118_x_at Vim 2618.58 intermediate filament 1456292_a_at Vim 2608.81 intermediate filament 1433472_x_at Rpl38 2580.65 skeletal system development/ossification/regulation of translation 90S preribosome assembly 1437341_x_at Cnp 2577.62 myelin associated enzyme 1435873_a_at Rpl13a 2577.15 transcription, DNA-dependent/regulation of translation 1418884_x_at Tuba1a 2550.18 microtubule formation 1423859_a_at Ptgds 2546.28 prostaglandin biosynthetic process 1451718_at Plp1 2532.85 axon ensheathment/myelination 1434670_at Kif5a 2531.08 microtubule-based transport 1425468_at Plp1 2493.37 axon ensheathment/myelination 1438859_x_at Rps29 2479.96 translation 1436201_x_at Mbp 2465.44 myelination 1418273_a_at Rpl30 2448.88 translation 1455439_a_at Lgals1 2432.62 cell adhesion 1455693_x_at Rps6-ps4 2427.72 G1/S transition of mitotic cell cycle 1460561_x_at Sepw1 2420.65 cell redox homeostasis 1416178_a_at Plekhb1 2420.34 synapse formation/axon guidance 1416496_at Mrfap1 2409.47 T-Cell Activation Protein 1460581_a_at Rpl13 2400.75 translation 1448344_at Rps12 2398.96 translation 1455138_x_at Cfl1 2372.17 actin PH-dependent polymerization/depolymerization 1419646_a_at Mbp 2369.59 myelination 1433991_x_at Dbi 2367.62 triglyceride metabolic process 1428212_x_at Rpl31-ps12 2360.56 translation 1438986_x_at Rps17 2357.14 ribosomal small subunit assembly/translation 1454651_x_at Mbp 2356.82 myelination 1455485_x_at Rpl13a 2355.27 transcription, DNA-dependent/regulation of translation 1436924_x_at Rpl31-ps12 2353.32 translation 1434872_x_at Rpl37 2333.66 translation 1454620_x_at Rps6-ps4 2330.06 G1/S transition of mitotic cell cycle 1415906_at Tmsb4x 2329.73 regulation of actin polymerization 1416453_x_at Rps12 2329.64 translation 1448392_at Sparc 2329.64 ossification 1437976_x_at Rpl23a 2318.12 translation/cell proliferation 1417275_at Mal 2313.86 myelination 1419734_at Actb 2311.81 actin formation 1437992_x_at Gja1 2311.46 gap junction formation 1426661_at Rpl27a 2310.13 translation 1454849_x_at Clu 2301.05 response to oxidative stress/cell death 1426195_a_at Cst3 2286.90 inhibitor of cysteine proteinases 1456174_x_at Ndrg1 2272.00 cellular response to hypoxia/stress 1450840_a_at Rpl39 2266.40 translation 1437666_x_at Ubc 2249.80 protein degradation 1443823_s_at Atp1a2 2241.20 cation transport ATPase 1415964_at Scd1 2231.29 lipid metabolic process 1438649_x_at Pebp1 2230.26 MAPK cascade/regulation of neurotransmitter levels/negative regulation of protein phosphorylation 1418209_a_at Pfn2 2225.22 cytoskeleton organization 1433443_a_at Hmgcs1 2221.52 ketogenesis/metabolism 1423860_at Ptgds 2219.19 prostaglandin biosynthetic process 1460008_x_at Rpl31-ps12 2216.88 translation 1437993_x_at Qdpr 2208.01 L-phenylalanine catabolic process 1416624_a_at Kxd1/Uba52 2195.34 translation 1456373_x_at Rps20 2188.32 translation 1452209_at Pkp4 2187.63 cell-cell junction assembly/cell adhesion 1451101_a_at Rps28 2182.10 rRNA export from nucleus/translation 1456228_x_at Mbp 2178.42 myelination 1434377_x_at Rps6-ps4 2172.54 G1/S transition of mitotic cell cycle 1425546_a_at Srprb 2156.02 GTPase 1438650_x_at Gja1 2139.54 gap junction formation 1416219_at Rpl19 2126.57 translation 1426660_x_at Rpl23a 2122.98 translation/cell proliferation 1438958_x_at Fkbp1a 2114.83 protein peptidyl-prolyl isomerization/negative regulation of protein phosphorylation 1438093_x_at Dbi 2103.06 triglyceride metabolic process 1415822_at Scd2 2100.66 lipid metabolic process/myelination 1416371_at Apod 2096.55 lipid metabolism 1448729_a_at Sept4 2093.88 filament-forming cytoskeletal GTPase 1451586_at Tmbim6 2092.92 anti apoptotic process/response to unfolded protein 1456436_x_at Rps20 2089.95 translation 1434369_a_at Cryab 2076.43 molecular chaperone
75 1416642_a_at Tpt1 2069.15 response to virus/stem cell maintenance 1455422_x_at Sept4 2063.26 filament-forming cytoskeletal GTPase 1436691_x_at Prdx1 2062.82 cell redox regulation 1456196_x_at Fkbp1a 2060.74 protein peptidyl-prolyl isomerization/negative regulation of protein phosphorylation 1427021_s_at Fth1 2057.13 iron ion transport/cellular iron ion homeostasis 1417463_a_at Lamtor1 2049.74 regulation of receptor recycling/endosome organization/lysosome organization 1433445_x_at Hmgcs1 2040.65 ketogenesis/metabolism 1425966_x_at Ubc 2040.17 protein degradation 1423091_a_at Gpm6b 2039.09 protein transport 1436991_x_at Gsn 2036.18 actin assembly and disassembly 1415716_a_at Rps27 2026.99 translation 1437729_at Rpl27a 2024.12 translation 1437171_x_at Gsn 2023.92 actin assembly and disassembly 1460175_at Rps23 2020.43 translation 1434892_x_at Rbbp4 2019.72 DNA replication/chromatin remodeling 1448157_s_at Rpl10 2018.71 translation 1437211_x_at Elovl5 2017.57 lipid metabolic process 1438655_a_at Rpl34-ps1 2017.39 translation 1448768_at Mog 2010.19 cell adhesion 1436064_x_at Rps24 2008.82 maturation of SSU-rRNA from tricistronic rRNA transcript/rRNA processing 1415849_s_at Stmn1 2004.60 microtubule depolymerization 1453723_x_at Ubb 1998.50 protein degradation 1438839_a_at Ywhae 1984.53 protein targeting/small GTPase mediated signal transduction 1424635_at Eef1a1 1953.51 translational elongation/protein homooligomerization 1436992_x_at Vdac1 1926.39 mitochondrial anion channel 1421375_a_at S100a6 1902.97 calcium binding
76 Table 3.4 Expression levels of the top 100 genes in 18-month optic nerves.
Probe Set ID Gene Symbol E Coding protein role/function
1435873_a_at Rpl13a 4013.13 transcription, DNA-dependent/regulation of translation 1455439_a_at Lgals1 4001.13 cell adhesion 1460581_a_at Rpl13 3866.90 translation 1448771_a_at Fth1 3865.98 iron ion transport and homeostasis 1433432_x_at Rps12 3799.24 translation 1418273_a_at Rpl30 3754.28 translation 1455976_x_at Dbi 3736.73 triglyceride metabolic process 1433472_x_at Rpl38 3736.10 skeletal system development/ossification/regulation of translation 90S preribosome assembly 1448344_at Rps12 3716.20 translation 1433532_a_at Mbp 3714.41 myelination 1438986_x_at Rps17 3706.89 ribosomal small subunit assembly/translation 1417184_s_at Hbb-bs/Hbb-bt 3687.28 hemoglobin 1438859_x_at Rps29 3682.26 translation 1433785_at Mobp 3668.36 intracellular protein transport/nervous system development 1416453_x_at Rps12 3659.59 translation 1455485_x_at Rpl13a 3604.31 transcription, DNA-dependent/regulation of translation 1436924_x_at Rpl31-ps12 3576.59 translation/protein folding 1437976_x_at Rpl23a 3525.91 translation/cell proliferation 1437726_x_at C1qb 3521.90 complement activation, classical pathway/innate immune response 1456292_a_at Vim 3487.19 intermediate filament 1438118_x_at Vim 3484.26 intermediate filament 1460008_x_at Rpl31-ps12 3478.28 translation/protein folding 1456373_x_at Rps20 3463.71 translation 1455693_x_at Rps6-ps4 3456.92 G1/S transition of mitotic cell cycle 1418884_x_at Tuba1a 3442.92 microtubule formation 1460561_x_at Sepw1 3439.24 cell redox homeostasis 1428212_x_at Rpl31-ps12 3439.02 translation 1438649_x_at Pebp1 3434.10 MAPK cascade/regulation of neurotransmitter levels/negative regulation of protein phosphorylation 1434872_x_at Rpl37 3430.83 translation 1450840_a_at Rpl39 3430.83 translation 1455245_x_at Rpl13 3411.19 translation 1416219_at Rpl19 3394.11 translation 1454620_x_at Rps6-ps4 3383.28 G1/S transition of mitotic cell cycle 1426661_at Rpl27a 3373.43 translation 1456436_x_at Rps20 3364.97 translation 1436996_x_at Lyz1 3360.50 immune system/cytolysis 1415906_at Tmsb4x 3355.44 regulation of actin polymerization 1423859_a_at Ptgds 3350.44 prostaglandin biosynthetic process 1433991_x_at Dbi 3339.14 triglyceride metabolic process 1438655_a_at Rpl34-ps1 3319.75 translation 1438093_x_at Dbi 3319.74 triglyceride metabolic process 1434231_x_at Rpl35 3316.48 translation 1426195_a_at Cst3 3308.23 inhibitor of cysteine proteinases 1460637_s_at Pfdn5 3294.99 protein folding 1460175_at Rps23 3294.82 translation 1415716_a_at Rps27 3291.18 translation 1451101_a_at Rps28 3286.67 rRNA export from nucleus/translation 1416970_a_at Cox7a2 3282.66 oxidation-reduction process 1436201_x_at Mbp 3264.41 myelination 1454856_x_at Rpl35 3264.39 translation 1448157_s_at Rpl10 3260.17 translation 1434670_at Kif5a 3258.49 microtubule-based transport 1449196_a_at Rps27a 3256.17 translation/ubiquitin homeostasis 1416178_a_at Plekhb1 3209.54 synapse formation/axon guidance 1437975_a_at Rpl23a 3199.25 translation/cell proliferation 1437729_at Rpl27a 3185.40 translation 1454627_a_at Rpl29 3167.92 translation/cell proliferation 1437510_x_at Rps17 3167.31 ribosomal small subunit assembly/translation 1416624_a_at Kxd1/Uba52 3157.99 translation 1449289_a_at B2m 3157.73 positive regulation of T cell mediated cytotoxicity/immune response 1452701_x_at Uba52 3154.04 translation 1426659_a_at Rpl23a 3149.88 translation/cell proliferation 1454651_x_at Mbp 3122.80 myelination 1416519_at Rpl36 3109.20 translation 1454778_x_at Rps28 3104.65 rRNA export from nucleus/translation 1436757_a_at Cox6b1 3099.89 oxidation-reduction process 1417317_s_at Rpl35a 3095.91 mitotic sister chromatid segregation/DNA repair/chromatin remodeling 1454849_x_at Clu 3094.87 response to oxidative stress/cell death 1422557_s_at Mt1 3084.14 melatonin 1436840_x_at Rpl35 3068.62 translation 1437341_x_at Cnp 3063.82 myelin associated enzyme 1426660_x_at Rpl23a 3051.40 translation/cell proliferation 1436064_x_at Rps24 3049.70 maturation of SSU-rRNA from tricistronic rRNA transcript/rRNA processing 1455138_x_at Cfl1 3049.26 actin PH-dependent polymerization/depolymerization
77 1451068_s_at Rps25 3049.08 ribosomal small subunit assembly 1455950_x_at Rpl35 3045.98 translation 1460590_s_at Ywhaq 3039.46 protein targeting/small GTPase mediated signal transduction 1423754_at Ifitm3 3018.19 interferon inducible protein/response to biotic stimulus 1439374_x_at Rps10 3018.02 ribosomal small subunit assembly 1423763_x_at Rps28 3013.49 rRNA export from nucleus/translation 1456628_x_at Rps24 3012.54 maturation of SSU-rRNA from tricistronic rRNA transcript/rRNA processing 1436691_x_at Prdx1 3011.81 cell redox regulation 1423860_at Ptgds 3003.08 prostaglandin biosynthetic process 1416056_a_at Ndufb11 2994.84 electron transport chain 1436822_x_at Rpl17 2985.86 cellular glucose homeostasis 1454859_a_at Rpl23 2977.48 translation 1434377_x_at Rps6-ps4 2975.12 G1/S transition of mitotic cell cycle 1416371_at Apod 2970.05 lipid metabolism 1416088_a_at Rps15 2965.07 ribosomal small subunit assembly 1435151_a_at Rps3 2941.60 translation/induction of apoptosis/response to DNA damage stimulus 1455767_x_at Rpl21 2926.72 translation 1437413_x_at Rps29 2921.03 translation 1416277_a_at Rplp1 2920.96 translational elongation 1435791_x_at Rpl17 2914.41 cellular glucose homeostasis 1416496_at Mrfap1 2913.15 T-Cell Activation Protein 1449436_s_at Ubb 2911.61 protein degradation 1425183_a_at Rpl4 2911.11 translation 1436995_a_at Rpl26 2906.62 rRNA processing/translation 1437610_x_at Rps8 2896.87 translational elongation 1455789_x_at Hspa8 2888.59 cell morphogenesis/ATP catabolic proces/transcription, DNA-dependent
78
Figure 3.5 Top 100 most expressed entities in P10-12, 1-month and 18-month mouse optic nerve, classified according to their coding protein biological function and expressed as percentage.
79 3.2.4 Most prominent genes enriched in the P10-12 mouse optic nerve
The three-way comparison of the top 100 genes in each age-group identified 60 that were specifically enriched in the P10-12 mouse optic nerve
(Figure 3.4), which is an indication of the functionally most important genes at this age. Their gene symbols, expression levels and coding protein role/function are listed in Table 3.5. The vast majority of these genes are involved in transcription, translation and protein folding, myelination, glial intermediate filaments and cytoskeleton organization and cellular metabolism. These are consistent with developmental myelination and growth at this age. Notable amongst the remaining genes was Sparc (Osteonectin), which was the most expressed and encodes for a cysteine-rich acidic matrix associated protein that is required for the collagen in bone to calcify and is involved in extracellular matrix synthesis (Brekken & Sage, 2001). High levels of Sparc mRNA are found in the developing and mature brain (Vincent et al., 2008; Eroglu, 2009); astrocyte- secreted SPARC can negatively regulate CNS synaptogenesis in vitro and in vivo and control synaptic AMPA receptor levels (Jones et al., 2011). Other notable genes include Bcas1 (Breast Cancer Amplified Sequence 1), which was the fourth most expressed and is associated with aggressive tumour phenotypes. In the brain, Bcas1 has been found to be regulated in the subthalamic nucleus of mice exposed to MPTP (Lauridsen et al., 2011). Col1a2 (Collagen type 1 alpha 2) is also enriched in the postnatal nerve and encodes for a fibril-forming collagen found in most connective tissue, and has been suggested in human neuroblastoma cultures as a marker for differentiation of these cells into glial cells in vitro
80 (DeClerck et al., 1987), but has not been directly reported in glia. Interestingly, there is high level of expression of Hba-a1/Hba-a2 (Haemoglobin alpha1/alpha2), iron-containing proteins associated with oxygen transport in the blood, which colocalise with mitochondria in the brain (Shephard et al., 2014).
81 Table 3.5 Most prominent genes enriched in the P10-12 mouse optic nerve
Probe Set ID Gene Symbol E Coding protein role/function 1416589_at Sparc 11961.59 ossification 1450641_at Vim 11353.20 intermediate filament 1422156_a_at Rps2 10235.47 ribosomal small subunit assembly/formation of translation preinitiation complex/translation 1428792_at Bcas1 9868.19 breast carcinoma-amplified sequence 1 1436722_a_at Actb 9831.44 actin formation 1451961_a_at Mbp 9680.36 myelination 1425467_a_at Plp1 9643.99 axon ensheathment/myelination 1432827_x_at Ubc 9643.52 protein degradation 1417451_a_at Ppia 9471.38 protein peptidyl-prolyl isomerization/protein folding 1431765_a_at Rps2 9436.73 ribosomal small subunit assembly/formation of translation preinitiation complex/translation 1415833_x_at Rps29 9400.98 translation 1426508_at Gfap 9327.45 intermediate filament 1422475_a_at Rps3a1 9264.44 translation 1415879_a_at Rplp2 9206.69 translational elongation 1449296_a_at Cnp 9188.18 microtubule cytoskeleton organization/axonogenesis 1455578_x_at Rpl41 9136.65 translation 1437185_s_at Tmsb10 9128.30 cytoskeleton organization 1450857_a_at Col1a2 9073.23 collagen formation 1415701_x_at Rpl23 9054.40 translation 1422613_a_at Rpl7a 9002.88 ribosome biogenesis 1428361_x_at Hba-a1/Hba-a2 8986.98 hemoglobin 1418625_s_at Gapdh 8882.26 glucose metabolic process/glycolysis 1459986_a_at Rps17 8857.92 ribosomal small subunit assembly/translation 1416141_a_at Rps6-ps4 8823.63 G1/S transition of mitotic cell cycle 1418000_a_at Itm2b 8790.52 apoptotic process/negative regulation of amyloid precursor protein biosynthetic process 1415812_at Gsn 8695.25 actin assembly and disassembly 1426088_at ND5 8688.92 electron transport chain 1416420_a_at Rpl9 8639.12 translation 1416330_at Cd81 8628.89 activation of MAPK activity 1436263_at Mobp 8570.69 intracellular protein transport/nervous system development 1436201_x_at Mbp 8496.67 myelination 1423507_a_at Sirt2 8421.87 protein deacetylation 1450088_a_at Mobp 8392.09 intracellular protein transport/nervous system development 1416404_s_at Rps16-ps2 8386.95 rRNA processing/translation 1455662_x_at Rps17 8365.08 ribosomal small subunit assembly/translation 1415942_at Rpl10 8361.41 translation 1423846_x_at Tuba1b 8352.84 microtubule formation 1448152_at Igf2 8313.95 development and growth 1449323_a_at Rpl3 8307.01 translation/cellular response to interleukin-4 1425742_a_at Tsc22d1 8286.70 TGF beta 1 induced trascriptional repressor 1455997_a_at Uqcrb 8283.02 mitochondrial electron transport 1437005_a_at Rpl18 8278.18 translation/oxidation-reduction process 1455106_a_at Ckb 8251.62 energy homeostasis 1435643_x_at Ubb 8231.31 protein degradation 1416099_at Rpl27 8224.69 translation 1432466_a_at Apoe 8209.40 lipid metabolism 1456142_x_at Morf4l1 8208.23 chromatin remodeling/transcriptional activation 1433721_x_at Rps21 8145.56 translation/ribosomal small subunit biogenesis 1423665_a_at Rpl5 8123.34 rRNA processing/translation 1437706_x_at Rps14 8119.01 ribosomal small subunit assembly/regulation of translation 1434396_a_at Myl6 8105.84 regulatory light chain of myosin 1428161_a_at Chchd2 8096.82 Coiled-Coil-Helix-Coiled-Coil-Helix Domain-Containing Protein 2 1416074_a_at Rpl28 8093.01 translation 1448739_x_at Rps18 8025.49 translation/ribosome biogenesis 1420830_x_at Ywhaq 8007.41 protein targeting/small GTPase mediated signal transduction 1419441_at Rplp0 7979.32 translational elongation/ribosome biogenesis/cellular response to interleukin-4 1454661_at Atp5g3 7974.93 ATP synthesis coupled proton transport 1423221_at Tubb4a 7969.45 microtubule formation
82 3.2.5 Most prominent genes enriched in the 1-month mouse optic nerve
The three-way comparison of the top 100 genes in each age-group identified 31 that were specifically enriched in the 1-month mouse optic nerve
(Figure 3.4), which is an indication of the functionally most important genes at this age. Their gene symbols, expression levels and coding protein role/function are listed in Table 3.6. The vast majority of these probes are for genes involved in glial intermediate filaments and cytoskeleton organization, control of cellular homeostasis and metabolism. One of the most expressed genes was Gja1 (Gap
Junction Protein alpha 1), which encodes for the main astroglial connexin Cx43; connexins are components of gap junctions, which are ubiquitous structures connecting cells, and are particularly abundant in CNS astrocytes (Dermietzel &
Spray, 1998). Ndrg1 (N-myc Downstream Regulated Gene 1) belongs to the alpha/beta hydrolase superfamily and encodes for a cytoplasmic protein involved in stress responses, hormone responses, cell growth and differentiation
(Melotte et al., 2010). Ndrg1 is upregulated with myelination of Schwann cells
(Heller et al., 2014) and its deficiency leads to progressive PNS demyelination in mice (Okuda et al., 2004). Pkp4 (Plakophilin-4) encodes for a member of the
Armadillo-like protein that regulates cell adhesion and cytoskeletal organization
(Keil et al., 2013); Pkp4 mRNA has been localized in primary astrocyte cultures
(Thomsen & Lade Nielsen, 2011). Tmbim6 (transmembrane BAX Inhibitor Motif
Containing 6) encodes for the protein BAX inhibitor 1 that acts as a suppressor of apoptosis and has been shown to modulate calcium homeostasis in mouse cortical cultures (Hunsberger et al., 2011).
83
Table 3.6 Most prominent genes enriched in the 1-month mouse optic nerve
Probe Set ID Gene Symbol E 1mo Coding protein role/function 1425468_at Plp1 2493.37 axon ensheathment/myelination 1448392_at Sparc 2329.64 ossification 1419734_at Actb 2311.81 actin formation 1437992_x_at Gja1 2311.46 gap junction formation 1456174_x_at Ndrg1 2272.00 cellular response to hypoxia/stress 1437666_x_at Ubc 2249.80 protein degradation 1443823_s_at Atp1a2 2241.20 cation transport ATPase 1415964_at Scd1 2231.29 lipid metabolic process 1418209_a_at Pfn2 2225.22 cytoskeleton organization 1437993_x_at Qdpr 2208.01 L-phenylalanine catabolic process 1452209_at Pkp4 2187.63 cell-cell junction assembly/cell adhesion 1425546_a_at Srprb 2156.02 GTPase 1438650_x_at Gja1 2139.54 gap junction formation 1438958_x_at Fkbp1a 2114.83 protein peptidyl-prolyl isomerization/negative regulation of protein phosphorylation 1448729_a_at Sept4 2093.88 filament-forming cytoskeletal GTPase 1451586_at Tmbim6 2092.92 anti apoptotic process/response to unfolded protein 1434369_a_at Cryab 2076.43 molecular chaperone 1455422_x_at Sept4 2063.26 filament-forming cytoskeletal GTPase 1456196_x_at Fkbp1a 2060.74 protein peptidyl-prolyl isomerization/negative regulation of protein phosphorylation 1427021_s_at Fth1 2057.13 iron ion transport/cellular iron ion homeostasis 1417463_a_at Lamtor1 2049.74 regulation of receptor recycling/endosome organization/lysosome organization 1433445_x_at Hmgcs1 2040.65 ketogenesis/metabolism 1425966_x_at Ubc 2040.17 protein degradation 1423091_a_at Gpm6b 2039.09 protein transport 1436991_x_at Gsn 2036.18 actin assembly and disassembly 1437171_x_at Gsn 2023.92 actin assembly and disassembly 1434892_x_at Rbbp4 2019.72 DNA replication/chromatin remodeling 1437211_x_at Elovl5 2017.57 lipid metabolic process 1448768_at Mog 2010.19 cell adhesion 1415849_s_at Stmn1 2004.60 microtubule depolymerization 1438839_a_at Ywhae 1984.53 protein targeting/small GTPase mediated signal transduction 1436992_x_at Vdac1 1926.39 mitochondrial anion channel 1421375_a_at S100a6 1902.97 calcium binding
84 3.2.6 Most prominent genes enriched in the 18-month mouse optic nerve
The three-way comparison of the top 100 genes in each age-group identified 31 that were specifically enriched in the 18-month mouse optic nerve
(Figure 3.4), which is an indication of the functionally most important genes at this age. Their gene symbols, expression levels and coding protein role/function are listed in Table 3.7. The majority of these probes are for genes involved in transcription, translation, protein folding and cellular metabolism. Moreover, the enrichment of genes involved in the immune response was specific to this group:
C1qb (Complement Component C1q, B chain) encodes a major constituent of the complement system and is expressed in rat striatal microglia (Pasinetti et al.,
1999), and in macrophage-like cells in the infarct core of a mouse model of cortical focal ischemia (Van Beek et al., 2000); lysozyme, encoded by Lyz1, is an enzyme with bacteriolytic function and is usually found in body fluids; B2m
(Beta-2-microglobulin) encodes a component of the major histocompatibility complex (MHC) class I, which is involved in the presentation of peptide antigens to the immune system; Ifitm3 (Interferon Induced Transmembrane Protein) has antiviral properties by disrupting intracellular cholesterol homeostasis. While there is no evidence supporting glial expression of Lyz1 and B2m, Ifitm3 is expressed by astrocytes, but its function in the CNS is still largely unknown (Ibi et al., 2013).
85 Table 3.7 Most prominent genes enriched in the 18-month mouse optic nerve
Probe Set ID Gene Symbol E 18mo Coding protein role/function 1437726_x_at C1qb 3521.90 complement activation, classical pathway/innate immune response 1455245_x_at Rpl13 3411.19 translation 1436996_x_at Lyz1 3360.50 immune system/cytolysis 1434231_x_at Rpl35 3316.48 translation 1460637_s_at Pfdn5 3294.99 protein folding 1416970_a_at Cox7a2 3282.66 oxidation-reduction process 1454856_x_at Rpl35 3264.39 translation 1437975_a_at Rpl23a 3199.25 translation/cell proliferation 1454627_a_at Rpl29 3167.92 translation/cell proliferation 1437510_x_at Rps17 3167.31 ribosomal small subunit assembly/translation 1449289_a_at B2m 3157.73 positive regulation of T cell mediated cytotoxicity/immune response 1452701_x_at Uba52 3154.04 translation 1426659_a_at Rpl23a 3149.88 translation/cell proliferation 1416519_at Rpl36 3109.20 translation 1436757_a_at Cox6b1 3099.89 oxidation-reduction process 1417317_s_at Rpl35a 3095.91 mitotic sister chromatid segregation/DNA repair/chromatin remodeling 1422557_s_at Mt1 3084.14 melatonin 1436840_x_at Rpl35 3068.62 translation 1451068_s_at Rps25 3049.08 ribosomal small subunit assembly 1455950_x_at Rpl35 3045.98 translation 1460590_s_at Ywhaq 3039.46 protein targeting/small GTPase mediated signal transduction 1423754_at Ifitm3 3018.19 interferon inducible protein/response to biotic stimulus 1439374_x_at Rps10 3018.02 ribosomal small subunit assembly 1456628_x_at Rps24 3012.54 maturation of SSU-rRNA from tricistronic rRNA transcript/rRNA processing 1416056_a_at Ndufb11 2994.84 electron transport chain 1436822_x_at Rpl17 2985.86 cellular glucose homeostasis 1416088_a_at Rps15 2965.07 ribosomal small subunit assembly 1455767_x_at Rpl21 2926.72 translation 1435791_x_at Rpl17 2914.41 cellular glucose homeostasis 1425183_a_at Rpl4 2911.11 translation 1437610_x_at Rps8 2896.87 translational elongation
86 3.2.7 Genes enriched in more than one age group
The list of genes enriched in P10-12 and 1-month old mouse optic nerve is given in Table 3.8 and clearly shows the importance of the myelination process at these two ages. In contrast, P10-12 and 18-month optic nerves share high levels of expression for genes involved in transcription and translation, protein degradation and oxygen transport (Table 3.9). One of the genes enriched in P10-
12 and 1-month old nerves is notable because it has not been reported in glia:
Tpt1 (Tumor Protein, Translationally Controlled 1) encodes for the protein fortilin, which regulates apoptosis and is over-expressed in cancer cells, and fortilin knock-out severely compromises the formation of neural tissue and is embryonically lethal (Koide et al., 2009).
Amongst the genes enriched in 1-month and 18-month mouse optic nerves, three that stand out because of their function are Lgals1, Plekhb1 and
Clu. Lgals1 (Lectin, Galactoside-Binding, Soluble 1) encodes for the protein galectin-1, which belongs to the family of beta-galactoside-binding proteins implicated in modulating cell-cell and cell-matrix interactions; a recent study suggests that galectin-1 might have a neuroprotective role (Qiao et al., 2013).
Plekhb1 (Pleckstrin homology Domain Containing Family B Member1) encodes
PHR1, protein required for proper localization of retinogeniculate projections during development (Culican et al., 2009); the fact that Plekhb1 is one of the most enriched genes in adult and ageing optic nerve suggests a persistent role in maintaining these projections. Clusterin, the protein encoded by Clu, is a
87 secreted chaperone suggested to be involved in several basic biological events, including cell death, tumor progression and neurodegenerative disorders; studies indicate an important role of clusterin in the pathogenesis of Alzheimer’s
Disease (Holtzman, 2004; Thambisetty et al., 2010), while, in the optic nerve, clusterin upregulation in astrocytes leads to complement activation following crush injury (Ohlsson et al., 2003).
Unsurprisingly, the genes enriched in all age groups are encoding proteins involved in essential biological functions, such as transcription and translation
(ribosomal subunits), myelin and cytoskeleton organization (MBP, actin), and cellular metabolism (Table 3.11). Notably, Pebp1 (phosphatidylethanolamine binding protein 1, also known as PBP) is a phospholipid binding protein and serine protease inhibitor, and to my knowledge there is only one report of its expression in the cytoplasm of oligodendrocytes, describing a role in the organization of phospholipids in the myelin sheath (Moore et al., 1996).
Table 3.8 Enriched genes common to P10-12 and 1-month mouse optic nerves
Probe Set ID Gene Symbol E P10-12 E 1mo Coding protein role/function 1456228_x_at Mbp 12737.34 2178.42 myelination 1424635_at Eef1a1 11042.74 1953.51 translational elongation/protein homooligomerization 1416642_a_at Tpt1 10366.71 2069.15 cell survival 1433443_a_at Hmgcs1 10271.65 2221.52 ketogenesis/metabolism 1451718_at Plp1 9257.40 2532.85 axon ensheathmen/myelination 1415822_at Scd2 9125.70 2100.66 lipid metabolic process/myelination 1419646_a_at Mbp 8754.42 2369.59 myelination 1417275_at Mal 8632.12 2313.86 myelination 1453723_x_at Ubb 8412.61 1998.50 protein degradation
88 Table 3.9 Enriched genes common to P10-12 and 18-month mouse optic nerves
Probe Set ID Gene Symbol E P10-12 E 18mo Coding protein role/function 1449436_s_at Ubb 10302.05 2911.61 protein degradation 1437413_x_at Rps29 10113.04 2921.03 translation 1417184_s_at Hbb-bs/Hbb-bt 9756.65 3687.28 hemoglobin 1436995_a_at Rpl26 8951.73 2906.62 rRNA processing/translation 1454859_a_at Rpl23 8907.35 2977.48 translation 1416277_a_at Rplp1 8833.91 2920.96 translational elongation 1423763_x_at Rps28 8489.38 3013.49 rRNA export from nucleus/translation 1455789_x_at Hspa8 8371.99 2888.59 cell morphogenesis/ATP catabolic process/transcription, DNA-dependent 1435151_a_at Rps3 8296.52 2941.60 translation/induction of apoptosis/response to DNA damage stimulus 1454778_x_at Rps28 8283.73 3104.65 rRNA export from nucleus/translation 1449196_a_at Rps27a 8187.12 3256.17 translation/ubiquitin homeostasis
Table 3.10 Enriched genes common to 1-month and 18-month mouse optic nerves
Probe Set ID Gene Symbol E 1mo E 18mo Coding protein role/function 1433432_x_at Rps12 2626.77 3799.24 translation 1438118_x_at Vim 2618.58 3484.26 intermediate filament 1456292_a_at Vim 2608.81 3487.19 intermediate filament 1435873_a_at Rpl13a 2577.15 4013.13 transcription, DNA-dependent/regulation of translation 1423859_a_at Ptgds 2546.28 3350.44 prostaglandin biosynthetic process 1434670_at Kif5a 2531.08 3258.49 microtubule-based transport 1418273_a_at Rpl30 2448.88 3754.28 translation 1455439_a_at Lgals1 2432.62 4001.13 cell adhesion 1455693_x_at Rps6-ps4 2427.72 3456.92 G1/S transition of mitotic cell cycle 1460561_x_at Sepw1 2420.65 3439.24 cell redox homeostasis 1416178_a_at Plekhb1 2420.34 3209.54 synapse formation/axon guidance 1416496_at Mrfap1 2409.47 2913.15 T-Cell Activation Protein 1460581_a_at Rpl13 2400.75 3866.90 translation 1448344_at Rps12 2398.96 3716.20 translation 1455138_x_at Cfl1 2372.17 3049.26 actin PH-dependent polymerization/depolymerization 1433991_x_at Dbi 2367.62 3339.14 triglyceride metabolic process 1438986_x_at Rps17 2357.14 3706.89 ribosomal small subunit assembly/translation 1434872_x_at Rpl37 2333.66 3430.83 translation 1454620_x_at Rps6-ps4 2330.06 3383.28 G1/S transition of mitotic cell cycle 1416453_x_at Rps12 2329.64 3659.59 translation 1437976_x_at Rpl23a 2318.12 3525.91 translation/cell proliferation 1426661_at Rpl27a 2310.13 3373.43 translation 1454849_x_at Clu 2301.05 3094.87 response to oxidative stress/cell death 1426195_a_at Cst3 2286.90 3308.23 inhibitor of cysteine proteinases 1456373_x_at Rps20 2188.32 3463.71 translation 1434377_x_at Rps6-ps4 2172.54 2975.12 G1/S transition of mitotic cell cycle 1426660_x_at Rpl23a 2122.98 3051.40 translation/cell proliferation 1438093_x_at Dbi 2103.06 3319.74 triglyceride metabolic process 1416371_at Apod 2096.55 2970.05 lipid metabolism 1456436_x_at Rps20 2089.95 3364.97 translation 1436691_x_at Prdx1 2062.82 3011.81 cell redox regulation 1415716_a_at Rps27 2026.99 3291.18 translation 1437729_at Rpl27a 2024.12 3185.40 translation 1448157_s_at Rpl10 2018.71 3260.17 translation 1438655_a_at Rpl34-ps1 2017.39 3319.75 translation
89 Table 3.11 Enriched genes common to P10-12, 1-month and 18-month mouse optic nerves
Probe Set ID Gene Symbol E P10-12 E 1mo E 18mo Coding protein role/function 1433785_at Mobp 10785.70 2947.40 3668.36 intracellular protein transport/nervous system development 1437341_x_at Cnp 10768.35 2577.62 3063.82 myelin associated enzyme 1433472_x_at Rpl38 10659.44 2580.65 3736.10 regulation of translation 90S preribosome assembly 1415906_at Tmsb4x 10137.10 2329.73 3355.44 regulation of actin polymerization 1460008_x_at Rpl31-ps12 9990.17 2216.88 3478.28 translation/ protein folding 1423860_at Ptgds 9805.28 2219.19 3003.08 prostaglandin biosynthetic process 1436924_x_at Rpl31-ps12 9698.18 2353.32 3576.59 translation/protein folding 1416624_a_at Kxd1/Uba52 9621.25 2195.34 3157.99 translation 1418884_x_at Tuba1a 9507.54 2550.18 3442.92 microtubule formation 1438859_x_at Rps29 9437.38 2479.96 3682.26 translation 1460175_at Rps23 9021.69 2020.43 3294.82 translation 1455976_x_at Dbi 8920.19 2647.00 3736.73 triglyceride metabolic process 1433532_a_at Mbp 8866.95 2989.54 3714.41 myelination 1454651_x_at Mbp 8635.56 2356.82 3122.80 myelination 1451101_a_at Rps28 8631.81 2182.10 3286.67 rRNA export from nucleus/translation 1438649_x_at Pebp1 8624.36 2230.26 3434.10 MAPK cascade/regulation of neurotransmitter levels/negative regulation of protein phosphorylation 1448771_a_at Fth1 8611.06 2939.68 3865.98 iron ion transport and homeostasis 1436201_x_at Mbp 8496.67 2465.44 3264.41 myelination 1450840_a_at Rpl39 8392.41 2266.40 3430.83 translation 1436064_x_at Rps24 8367.11 2008.82 3049.70 maturation of SSU-rRNA from tricistronic rRNA transcript/rRNA processing 1455485_x_at Rpl13a 8280.98 2355.27 3604.31 transcription, DNA-dependent/regulation of translation 1428212_x_at Rpl31-ps12 8038.42 2360.56 3439.02 translation 1416219_at Rpl19 7980.54 2126.57 3394.11 translation
90 3.2.8 “Neurotransmission machinery” in young, adult and old optic nerve
There is abundant evidence of neurotransmission in the optic nerve and glial cells express an array of neurotransmitter receptors and have the capacity to synthesize and release neurotransmitters (Butt et al., 2014b). However, most studies have focused on the developing optic nerve, and little is known about age-related changes in the various aspects of the ‘neurotransmission machinery’.
To address this, I analysed 516 genes involved in cell-to-cell signaling, neurotransmitter production, storage and release, and filtered for an absolute expression value, E, 50 and displaying a fold change (FC) 1.5 between at least
2 age groups (Figure 3.6). In this way 230 genes of interest were identified and are presented in the following sections according to the official IUPHAR/BPS target classification (http://www.guidetopharmacology.org).
91
Figure 3.6 Venn diagram showing the number of entities for neurotransmission in filtered for E50 and FC 1.5 between at least 2 age groups
92 3.2.9 Class A G-protein coupled receptors (GPCRA) in the mouse optic nerve
The expression of genes for GPCRA is summarized in Table 3.12. Most of the GPCRA are called absent or expressed at barely detectable levels, and the most evident feature is a general downregulation of GPCRA after postnatal development (Figure 3.7). Most prominent in the optic nerve are the adenosine receptor Adora1 and purine receptor P2yr12. Only 13 genes are altered in expression by FC 1.5 between at least 2 age groups: Adora2a, Adora2b,
Adra1b, Adra2a, Adrb2, Chrm1 are detectable in P10-12 optic nerve only, suggesting that adrenergic and cholinergic receptors have a role in the development of young tissue. In addition, there is an apparent developmental switch in expression of 5-HT receptors, in particular Htr1b, which is enriched in postnatal nerves. The expression profile of purinergic receptors indicates a pre- eminence for P2ry12, which is highly expressed at P10-12 and then downregulated, but persists in 1-month and 18-month nerves, whilst P2ry13 shows an antithetical expression profile, with the highest expression detected in the ageing optic nerve.
93 Table 3.12 GPCRA in P10-12, 1-month and 18-month mouse optic nerve. Downregulation with age is depicted in red.
Gene Symbol P10-12 1mo 18mo FC P10-12 vs 1mo FC 1mo vs 18mo Official IUPHAR receptor name E Call E Call E Call Adora1 377.64 P 181.05 P 143.09 P 2.09 1.27 A1 receptor Adora2a 171.25 P 31.61 A 29.46 A 5.42 1.07 A2A receptor Adora2b 68.73 P 10.55 A 11.40 A 6.52 0.93 A2B receptor Adora3 8.27 A 3.08 A 4.81 A 2.68 0.64 A3 receptor Adra1a 4.53 A 2.57 A 6.18 A 1.76 0.42 α1A-adrenoceptor Adra1b 82.85 P 7.31 A 6.84 A 11.33 1.07 α1B-adrenoceptor Adra2a 207.85 P 30.04 A 16.39 A 6.92 1.83 α2A-adrenoceptor Adra2b 12.83 A 5.34 A 3.81 A 2.40 1.40 α2B-adrenoceptor Adra2c 21.64 A 2.40 A 2.76 A 9.01 0.87 α2C-adrenoceptor Adrb1 17.17 A 7.96 A 7.31 A 2.16 1.09 β1-adrenoceptor Adrb2 104.20 P 30.95 A 6.62 A 3.37 4.68 β2-adrenoceptor Adrb3 1.51 A 1.89 A 6.06 A 0.80 0.31 β3-adrenoceptor Chrm1 73.20 P 7.84 A 14.84 A 9.34 0.53 M1 receptor Chrm3 5.13 A 1.07 A 1.00 A 4.81 1.07 M3 receptor Chrm4 1.00 A 1.00 A 1.00 A 1.00 1.00 M4 receptor Cnr1 32.90 A 4.09 A 10.53 A 8.05 0.39 CB1 receptor Cnr2 7.75 A 5.71 A 13.75 A 1.36 0.42 CB2 receptor Drd1a 5.34 A 3.27 A 2.93 A 1.63 1.12 D1 receptor Drd2 16.40 A 1.40 A 4.99 A 11.71 0.28 D2 receptor Drd3 9.34 A 1.18 A 1.27 A 7.89 0.93 D3 receptor Drd4 13.37 A 4.52 A 3.40 A 2.96 1.33 D4 receptor Hrh1 28.69 A 8.42 A 22.97 A 3.41 0.37 H1 receptor Hrh2 15.72 A 3.51 A 3.95 A 4.48 0.89 H2 receptor Hrh3 12.52 A 1.84 A 6.55 A 6.82 0.28 H3 receptor Hrh4 1.40 A 1.00 A 1.74 A 1.40 0.57 H4 receptor Htr1a 21.34 A 1.53 A 3.43 A 13.93 0.45 5-HT1A receptor Htr1b 115.68 P 8.77 A 5.92 A 13.19 1.48 5-HT1B receptor Htr1d 11.62 A 3.65 A 3.84 A 3.18 0.95 5-HT1D receptor Htr1f 1.24 A 1.22 A 6.16 A 1.01 0.20 5-HT1F receptor Htr2b 6.26 A 75.34 P 57.78 P 0.08 1.30 5-HT2B receptor Htr2c 22.19 A 1.07 A 5.54 A 20.68 0.19 5-HT2C receptor Htr4 6.62 A 1.38 A 1.00 A 4.80 1.38 5-HT4 receptor Htr5a 8.87 A 1.59 A 4.94 A 5.59 0.32 5-HT5a receptor Htr5b 12.59 A 1.48 A 1.64 A 8.51 0.91 5-HT5b receptor Htr6 13.09 A 2.54 A 2.19 A 5.15 1.16 5-HT6 receptor Htr7 11.01 A 1.64 A 2.74 A 6.71 0.60 5-HT7 receptor Oprd1 2.76 A 1.05 A 1.00 A 2.62 1.05 δ receptor Oprk1 22.69 A 1.08 A 1.56 A 20.94 0.70 κ receptor Oprl1 41.70 A 2.41 A 4.89 A 17.32 0.49 NOP receptor Oprm1 8.29 A 1.30 A 2.27 A 6.36 0.58 μ receptor P2ry1 20.66 A 14.58 A 10.94 A 1.42 1.33 P2Y1 receptor P2ry12 928.45 P 148.44 P 108.84 P 6.25 1.36 P2Y12 receptor P2ry13 281.07 P 208.78 P 474.68 P 1.35 0.44 P2Y13 receptor P2ry14 67.31 P 54.79 P 83.56 P 1.23 0.66 P2Y14 receptor P2ry2 1.91 A 2.01 A 2.82 A 0.95 0.71 P2Y2 receptor P2ry4 24.24 A 1.20 A 1.00 A 20.22 1.20 P2Y4 receptor P2ry6 210.28 P 52.93 P 141.49 P 3.97 0.37 P2Y6 receptor
94
Absolute value Absolute (E)
Figure 3.7 Age-related changes of the most prominent GPCRA in the mouse optic nerve.
95
3.2.10 Class C G -protein-coupled receptors (GPCRC) in the mouse optic nerve
The expression of genes for GPCRC is summarized in Table 3.13. Most of the GPCRC are called absent or expressed at barely detectable levels, and the most evident feature is a general downregulation of GPCRC after postnatal development (Figure 3.8). Most prominent is the calcium sensing receptor CASR, which appears to be expressed only in the postnatal optic nerve. The metabotropic GABAergic GABAB receptor is highly expressed in all 3 age groups, suggesting a prominent and persistent role for GABA in the optic nerve. The metabotropic glutamate receptors (mGluR) show a varied expression profile, with Grm4 being the most expressed subunit at P10-12 and then it is downregulated, whereas Grm5 is most highly expressed in the ageing optic nerve.
96 Table 3.13 GPCRC in P10-12, 1-month and 18-month mouse optic nerve. Downregulation with age is depicted in red.
Gene Symbol ( GPPC1R0 C-1 )2 1mo 18mo FC P10-12 vs 1mo FC 1mo vs 18mo Official IUPHAR receptor name E Call E Call E Call Casr 93.81 P 2.21 A 1.00 A 42.48 2.21 CASR Gabbr1 409.90 P 350.12 P 332.48 P 1.17 1.05 GABAB1 Gprc6a 3.94 A 1.48 A 1.00 A 2.65 1.48 GPRC6 receptor Grm1 26.04 A 4.90 A 27.53 A 5.31 5.61 mGlu1 receptor Grm2 14.56 A 2.28 A 24.34 A 6.38 10.66 mGlu2 receptor Grm3 78.49 P 91.32 P 88.21 P 1.16 1.04 mGlu3 receptor Grm4 235.43 P 7.70 A 43.71 A 30.58 5.68 mGlu4 receptor Grm5 47.50 A 33.61 A 86.77 P 1.41 2.58 mGlu5 receptor Grm7 26.24 A 17.92 A 41.68 A 1.46 2.33 mGlu7 receptor Grm8 13.05 A 2.14 A 16.51 A 6.09 7.70 mGlu8 receptor
Absolute value Absolute (E)
Figure 3.8 Age-related changes of the most prominent GPCRA in the mouse optic nerve.
97 3.2.11 Expression of ligand-gated ion channels in the mouse optic nerve
The expression of ligand-gated ion channels (LGIC) is summarized in Table
3.14. Many LGIC are called absent or expressed at barely detectable levels, but a prominent feature is the expression of multiple iGluR and P2XR in postnatal nerves, whereas nAChR are not prominently expressed at any age, and
GABAA1/3, GABAA2 and GABA2 are upregulated in the 18-month nerve, whereas others are more prominent in the postnatal nerve (Figure 3.9). The gene for the glycine receptor is one the most expressed in all 3 groups. The most expressed genes in the postnatal nerve encode the AMPA receptor subunit
GluA1, whose mRNA level is downregulated but still high in the adult and ageing nerves. Kainate receptor genes (Grik1-3 and 5) appear to be most expressed, even though at low levels and downregulated after postnatal development, except for Grik5. The NMDA receptor subunit GluN2B appears to be expressed only in the old optic nerve. P2X4 and P2X7 are the purinergic receptors, whose mRNA are expressed the most at P10-12, and are downregulated thereafter.
98 Table 3.14 LGIC in P10-12, 1-month and 18-month mouse optic nerve. Downregulation with age is depicted in red.
Gene Symbol P10-12 1mo 18mo FC P10-12 vs 1mo FC 1mo vs 18mo Official IUPHAR receptor name E Call E Call E Call Chrna1 5.90 A 1.00 A 1.00 A 5.90 1.00 α1 Chrna2 7.03 A 1.00 A 2.52 A 7.03 2.52 α2 Chrna3 9.80 A 1.95 A 4.70 A 5.02 2.41 α3 Chrna4 38.75 A 8.30 A 6.90 A 4.67 1.20 α4 Chrna5 14.51 A 3.03 A 1.46 A 4.79 2.07 α5 Chrna6 6.04 A 2.60 A 4.67 A 2.32 1.79 α6 Chrna7 13.38 A 3.25 A 3.74 A 4.12 1.15 α7 Chrna9 3.74 A 1.94 A 1.75 A 1.93 1.11 α9 Chrnb1 53.58 P 49.17 A 14.64 A 1.09 3.36 β1 Chrnb2 20.57 A 3.48 A 14.95 A 5.91 4.30 β2 Chrnb3 3.94 A 1.66 A 2.03 A 2.38 1.23 β3 Chrnb4 13.77 A 1.31 A 4.90 A 10.50 3.74 β4 Chrnd 2.47 A 1.00 A 1.00 A 2.47 1.00 δ Chrne 14.06 A 2.92 A 1.00 A 4.82 2.92 ε Chrng 6.88 A 1.07 A 1.00 A 6.41 1.07 γ Gabra1 21.18 A 4.84 A 182.36 P 4.37 37.65 α1 Gabra2 15.80 A 2.78 A 43.16 A 5.67 15.50 α2 Gabra3 35.89 A 22.97 A 138.97 P 1.56 6.05 α3 Gabra4 37.73 A 7.31 A 32.36 A 5.16 4.43 α4 Gabra5 10.48 A 2.69 A 111.87 P 3.90 41.62 α5 Gabra6 6.92 A 1.12 A 1.51 A 6.18 1.35 α6 Gabrb1 208.34 P 246.72 P 402.99 P 0.84 1.63 β1 Gabrb2 30.28 A 16.66 A 185.11 P 1.82 11.11 β2 Gabrb3 165.60 P 32.11 A 84.40 P 5.16 2.63 β3 Gabrd 14.29 A 3.60 A 20.44 A 3.97 5.69 δ Gabre 6.78 A 1.32 A 1.92 A 5.15 1.46 ε Gabrg1 69.54 A 7.55 A 13.36 A 9.21 1.77 γ1 Gabrg2 23.24 A 2.30 A 103.48 P 10.12 45.06 γ2 Gabrg3 26.58 A 47.15 A 17.92 A 1.77 2.63 γ3 Gabrp 21.00 A 1.37 A 1.56 A 15.39 1.15 π Gabrq 2.59 A 1.00 A 1.00 A 2.59 1.00 θ Gabrr1 2.63 A 1.00 A 1.00 A 2.63 1.00 ρ1 Gabrr2 6.72 A 41.36 A 5.68 A 6.15 7.28 ρ2 Glra1 8.86 A 1.27 A 1.50 A 6.96 1.18 α1 Glra2 8.12 A 1.76 A 8.12 A 4.62 4.62 α2 Glra3 1.01 A 1.00 A 1.97 A 1.01 1.96 α3 Glra4 1.04 A 1.00 A 1.00 A 1.04 1.00 α4 Glrb 435.84 P 405.05 P 405.30 P 1.08 1.00 β Gria1 1185.38 P 496.67 P 528.29 P 2.39 1.06 GluA1 Gria2 248.16 P 59.95 P 208.64 P 4.14 3.48 GluA2 Gria3 79.38 P 47.63 A 87.95 P 1.67 1.85 GluA3 Gria4 254.42 P 91.87 P 70.05 P 2.77 1.31 GluA4 Grid1 43.16 A 14.72 A 11.51 A 2.93 1.28 GluD1 Grid2 96.82 A 31.52 A 9.89 A 3.07 3.19 GluD2 Grik1 17.91 A 103.96 A 38.05 A 5.80 2.73 GluK1 Grik2 68.58 P 53.41 P 45.32 A 1.28 1.18 GluK2 Grik3 53.97 A 14.61 A 21.20 A 3.69 1.45 GluK3 Grik4 48.61 A 26.93 A 17.85 A 1.81 1.51 GluK4 Grik5 387.40 A 28.77 A 32.59 A 13.46 1.13 GluK5 Grin1 65.66 A 5.98 A 49.95 A 10.98 8.35 GluN1 Grin2a 1.00 A 1.00 A 1.00 A 1.00 1.00 GluN2A Grin2b 20.45 A 4.64 A 116.94 P 4.41 25.21 GluN2B Grin2c 2.34 A 1.06 A 22.81 A 2.20 21.49 GluN2C Grin2d 54.42 A 23.80 A 40.60 A 2.29 1.71 GluN2D Grin3a 95.39 A 22.11 A 19.76 A 4.31 1.12 GluN3A Grin3b 6.11 A 12.23 A 13.89 A 0.50 1.14 GluN3B Htr3a 26.91 A 2.17 A 5.26 A 12.40 2.42 5-HT3A Htr3b 1.00 A 1.18 A 1.00 A 0.85 1.18 5-HT3B P2rx1 20.29 A 2.92 A 2.13 A 6.96 1.37 P2X1 P2rx2 2.43 A 1.16 A 1.00 A 2.10 1.16 P2X2 P2rx3 2.37 A 3.70 A 4.16 A 0.64 1.12 P2X3 P2rx4 311.92 P 89.09 P 74.63 P 3.50 1.19 P2X4 P2rx5 11.84 A 5.71 A 4.98 A 2.07 1.15 P2X5 P2rx6 46.38 A 31.94 A 27.42 A 1.45 1.16 P2X6 P2rx7 178.53 P 115.55 P 51.92 P 1.55 2.23 P2X7
99
value Absolute (E)
Figure 3.9 Age-related changes of the most prominent LGIC in the mouse optic nerve.
0 10
3.2.12 Expression of genes involved in neurotransmission in the mouse optic
nerve
The expression of genes that are essential for neurotransmission were examined in brief and are summarized in Figures 3.10-3.12, and the key point is that the optic nerve contains the machinery for neurotransmission, and in general this is downregulated with age. The neurotransmitter transporters, enzymes and SNAREs are summarized in Figure 3.10, and the most evident feature is that neurotransmitter transporters are expressed at their highest level in the postnatal optic nerve. Slc1a3 is the most highly expressed gene and encodes EAAT1/GLAST, the main astrocytic glutamate transporter. The genes for glutamate and GABA transporters (Slc1a1-3 and Slc6a1/6/8/11/12/13) are highly expressed at P10-12 and then are downregulated. On the other hand, the glycine transporter GlyT1 (Slc6a9) is prominently expressed at all ages. The vesicular glutamate and ATP transporters are not evident, although VGLUT1 (Slcc17a7) appears to be expressed in the 18-month nerve. Slc18b1 is a paralog of Slc18a1, which encodes vesicular monoamine transporter 1 (VMAT1), and is expressed at all ages.
The expression of enzymes associated with neurotransmission is summarized in Figure 3.11. Notably Adk (adenosine kinase) was prominent at all ages and encodes for an enzyme that catalyzes ATP to adenosine, both intracellularly and extracellularly, consistent with the prominent role for ATP- mediated signaling in the optic nerve (Butt, 2011). In addition, Aldh9a1
101 (Aldehyde Dehydrogenase 9, member A1) and Aldh5a1 (Aldehyde
Dehydrogenase 5, member A1) are expressed at all ages and important in the breakdown of GABA, whereas Gad 1 and Gad2 (glutamate decarboxylase 1 and
2) were most highly expressed in the ageing nerve and convert GABA to glutamate, a function of astrocytes. Notably, Mg11 (Monoglyceride lipase) is highly expressed in the postnatal nerve, and Gaglb (Diaglycerol Lipase, beta) appears to be upregulated with age, both playing a critical role in the formation of the endocannabinoid 2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid in tissues.
The expression of the most prominent genes that encode synaptic proteins is summarized in Figure 3.12, which suggests highly active synaptic transmission in the optic nerve. Notable amongst these were the high levels of transcripts for Vamp3 (Vesicle associated membrane protein 3, or synaptobrevin-3) and Vapa (Vesicle associated protein A), which functions are related to vesicle trafficking, docking and fusion, and were highest in the postnatal nerve, suggesting they may be primarily axonal (Alix et al., 2008).
Vesicular release in astrocytes usually involves synaptobrevin-2 (Vamp2), which was present at all ages in the optic nerve, and snap-23, which was most prominent in the postnatal optic nerve. Syntaxins were also prominent, with an apparent developmental shift from syntaxin 6/7/16 (Stx6/7/16) in postnatal nerves to syntaxin 12 (Stx12) in ageing nerves; the syntaxins are part of the
SNARE complex that also contains VAMPs and regulate synaptic vesicles and endosomes, a feature of astrocyte neurotransmitter release.
102
Absolute value Absolute (E)
Figure 3.10 Age-related changes of the most prominent neurotransmitter transporters in the mouse optic nerve.
3 10
value Absolute (E)
Figure 3.11 Age-related changes of the most prominent enzymes involved in neurotransmission in the mouse optic nerve.
4 10
Absolute value Absolute (E)
Figure 3.12 Age-related changes of the most prominent SNARE and synaptic proteins involved in neurotransmission in the mouse optic nerve. 5 10
3.3 Discussion
Astrocytes and oligodendrocytes have been shown to express a wide range of neurotransmitter receptors in vitro and in vivo (Verkhratskiĭ &
Butt, 2013). Furthermore, there is abundant evidence that neurotransmitter- mediated signalling is a prominent feature of CNS white matter, even though it does not contain neuronal cell bodies, dendrites or synapses (see (Butt et al.,
2014a) for recent review). In this section, I have provided a profile of neurotransmitter signalling in the optic nerve and how it changes with age
(Figure 3.13). Notably, the optic nerve is enriched in the machinery for neurotransmission, with a prominence for glutamate > ATP > GABA > Glycine >
Adenosine > Adrenergic. A key finding is that neurotransmitter systems were highest in the postnatal nerve, supporting evidence of important roles in oligodendrocyte differentiation and myelination. Some notable exceptions were
GABA signalling, mGluR5 and P2Y13 receptors, which were increased in the 18- month optic nerve and hence may be important in the neuropathology of white matter loss, which is a feature of the ageing brain and dementia. To this end, I also examined key oligodendrocyte genes and the main change in the ageing optic nerve was a decrease in OPC genes and transcription factors that regulate their differentiation, suggesting OPC function may be deregulated in ageing white matter. In addition, my analysis identified a number of genes that have no reported function in CNS glia and were specifically enriched in the ageing optic nerve. This study helps to shed new light on the key neurotransmitter systems
106 and potential molecular and cellular targets that may be important in regulating changes in glial functions with age.
107
Figure 3.13 Summary of age-related changes in the main neurotransmitter receptors in the mouse optic nerve.
108 3.3.1 Age-dependent changes in glial genes
Microarray analysis confirmed that the bulk of gene expressed in the optic nerve at all ages is specific to astrocytes and oligodendrocytes, which make up the vast majority of cells in the optic nerve, with relatively lower levels of expression of OPC, microglia, vascular and axonal genes (Salter & Fern, 2005). A three-way analysis in postnatal, adult and ageing optic nerve shows that the most highly expressed genes are involved in the primary glial functions of myelination, homeostasis and metabolism, in addition to cell growth, e.g. cytoskeleton and intermediate filament formation and protein degradation. A key finding is the decline in genes associated with myelination, homeostasis/metabolism and cell growth. My analysis highlighted a reduction of oligodendrocyte and OPCs markers from postnatal to adult and further still in ageing tissue (Figure 3.14). As predicted, the P10-12 optic nerves is enriched in myelin and OPC genes, since it is undergoing highly active myelination, which commences around P7 and the majority of axons are myelinated by P15 (Foran &
Peterson, 1992). Notably, compared to the adult nerve at 1-month, there was an apparent loss of the OPC marker Pdgfra in the aging 18-month optic nerve, as well as a striking decrease in transcription factors that regulate OPC differentiation, Nkx2.2, Sox10 and Olig2 (Figure 3.14B), which are essential for initiating oligodendrocyte differentiation (Zhu et al., 2014). The decline in OPCs regenerative capability in the ageing brain has been suggested to be a key factor in the age-dependent loss of white matter and decline in cognitive function (Sim et al., 2002; Bartzokis, 2011a; b). The results of my study indicate this may be
109 related to a loss of PDGF-mediated signalling, which regulates OPC proliferation, and key transcription factors that regulate their subsequent differentiation into oligodendrocytes.
In contrast to the decline in genes regulating myelination, there is a marked increase in genes associated with transcription/translation/protein folding in the 18-month optic nerve, indicative of altered proteostasis which is associated with stress and ageing (Hipp et al., 2014). The maintenance of the proteome is regulated by a complex proteostasis network that is essential to preserve cell function and the ability to respond and adapt to the changing environment (Hipp et al., 2014). My results in the ageing optic nerve require further investigation, but may identify genes and/or pathways that underlie the decline in protein homeostasis that is a feature of ageing and is likely to contribute to white matter disruption during ageing.
110
Figure 3.14 Age-related changes in the main oligodendrocyte and OPC genes in the mouse optic nerve.
111 3.3.2 Microglial genes and ageing mechanisms in the optic nerve
Although only a small number of microglial genes were examined, proportionally they comprised 3% of genes in the postnatal and adult nerve, which is consistent with number of microglia in relation to astrocytes and oligodendrocytes. Interestingly, the microglial markers Mrc1 (CD206) and Fcgr3
(CD16) exhibited opposite developmental shifts, with Mrc1 being high in the postnatal nerve and CD16/FcR3 being increased in the ageing optic nerve. Both
Mrc1 and Fcgr3 are considered markers of the M2 phenotype (Kigerl et al.,
2009), and in my analysis there did not appear to be a clear-cut shift between the M1 (activated, or pro-inflammatory) and M2 (alternatively activated or anti- inflammatory, involved in restoring tissue homeostasis) phenotypes. The M1 phenotype, or classical activation, is associated with defence and attack and induces the release of pro-inflammatory cytokines (Mantovani et al., 2004;
Mosser & Edwards, 2008). The M2 phenotypes are termed alternative activation and include the M2a, M2b and M2c states. The M2a phenotype is characterized by wound healing and high levels of arginase 1 (ARG1) and the chitinase-like protein YM1 (Edwards et al., 2006). The M2b phenotype is a combination of an
M1 and M2a state and is associated with high levels of CD86 and the Fc gamma receptors 1 and 3 (FcγR1 and FcγR3) (Edwards et al., 2006). Finally, the M2c phenotype is a deactivation state accompanied by high levels of transforming growth factor beta and sphingosine kinase 1 (Sternberg, 2006; Mosser &
Edwards, 2008).
112 Senescent CD16+ macrophages also exhibit proinflammatory activity and their accumulation could have a role in the development of chronic inflammation and atherosclerosis in elderly subjects (Merino et al., 2011). Moreover, a transition from M1 to a mixed phenotype increases amyloid deposition in a double transgenic model of AD (Weekman et al., 2014). In the 18-month optic nerve there was an apparent enrichment in genes involved in the innate immunity, most notably C1qb (Complement Component C1q, B chain) and lysozyme, encoded by Lyz1, the MHCI co-factor B2m (Beta-2-microglobulin), and
Ifitm3 (Interferon Induced Transmembrane Protein). MHCI is localized on all nucleated cells and the MHCI cofactor B2m has been shown to be upregulated in the hippocampus with advanced aging (Vanguilder et al., 2012). C1qb encodes a major constituent of the complement system in microglia (Pasinetti et al., 1999), which is increased in response to axonal injury (Byrnes et al., 2006) and in AD
(Johnson et al., 1992). Lysozyme is important in inflammasome activation in macrophages, and indicates increased phagocytic activity is microglia/macrophages in the ageing optic nerve. In contrast, upregulation of
Ifitm3 in astrocytes mediates neuronal impairments (Ibi et al., 2013) and it is implicated in the immune disturbance in schizophrenia and autism (Garbett et al., 2008a; Garbett et al., 2008b). Overall, the results suggest that multiple genes may contribute to altered immune homeostasis and white matter disruption in the ageing optic nerve.
113 3.3.3 Age-dependent changes in neurotransmitter receptors in the optic
nerve
The analysis on neurotransmitter receptors provides evidence for the presence of diverse signalling in white matter, primarily glutamatergic and purinergic systems, which have been extensively described, but also GABAergic, adrenergic, cholinergic, dopaminergic, serotoninergic and glycinergic signalling mechanisms were all detected, although their function is unclear (Figure 3.15).
Neurotransmitter receptors were most highly expressed during development, but the main ones continued to be expressed in the adult and ageing nerve.
However, adenosine receptors 2A (Adora2a) and 2B (Adora2b), the 2, 1, and
2 adrenergic receptors (Adra1b, Adra2a, Adrb2), and mAChR M1 (Chrm1) were detectable in P10-12 optic nerve only, suggesting they have a specific role in optic nerve development. Most of these neurotransmitters have been shown to affect OPCs and their differentiation in one manner or other, e.g. adenosine acting via A1 receptors regulates the migration, proliferation and differentiation of OPCs (Othman et al., 2003; Agresti et al., 2005a), and mAChR activation significantly increases OPC proliferation and inhibits their differentiation into myelinating oligodendrocytes (De Angelis et al.). In comparison, GABA signalling was highest in the ageing nerve and inhibits OPC proliferation (Yuan et al., 1998).
Interestingly, transcripts for glutamate receptors predominated by far in the postnatal optic nerve, whereas P2Y and P2XR are always most dominant in functional studies using calcium imaging (Hamilton et al., 2008; Hamilton et al.,
2010). This illustrates that glutamate receptors, like GABA receptors, do not play
114 a major role in mediating calcium signalling in optic nerve glia, but instead have other effects, such as sodium movement and intracellular signalling through second messengers other than calcium (Verkhratsky and Butt, 2013).
Transcripts for AMPA, Kainate and NMDA receptors are present in the optic nerve, in agreement with their expression by astrocytes and oligodendrocytes (Matute et al., 2007a; Bakiri et al., 2009; Matute, 2011). AMPA receptors are composed of GluRA1-4 subunits, and all four were detected at all ages analysed, although GluA1 and GluA4 were higher in the postnatal nerve, consistent with evidence that AMPA receptors help control the timing of myelination, by inhibiting OPC proliferation and differentiation (reviewed in
(Mangin & Gallo, 2011)). Interestingly, OPCs and myelin express NMDA-R that lack the NR2 subunit, which bears the glutamate binding site and instead they are activated exclusively by glycine or D-serine, and they display reduced Ca2+ permeability and Mg2+ sensitivity (reviewed in (Stys, 2011)). In contrast, transcripts in the optic nerve would indicate an upregulation of GluN2B in the ageing optic nerve, suggesting there may be a change in NMDA-R function to increased sensitivity to glutamate and Ca2+ permeability, which may have pathological consequences (Salter & Fern, 2005). The GluN2B subunit is reduced with age in hippocampus and cerebral cortex (Low & Wee, 2010), and when enhanced in aged mice can rescue memory (Brim et al., 2013). The high level of expression observed in the ageing optic nerve could be related to its reported
115 expression in astrocytes after ischaemia and anoxia in vivo and in vitro (Krebs et al., 2003).
Figure 3.15 Age-related changes in the neurotransmitter receptor expression in the mouse optic nerve.
116 Metabotropic glutamate receptors (mGluRs) were also prominent in the optic nerve, with the mGluR5 (group I), mGluR3 (group II), and mGluR4 (group III) being the most expressed. Expression of mGluR5 and mGluR3 is a common feature of glial cells throughout the CNS and they regulate multiple functions, such as inhibition of K+ currents, proliferation and survival (Verkhratsky and Butt,
2013). In contrast, mGluR4 is surprisingly the most expressed mGluR transcript in the postnatal optic nerve, although it has been described in cultured astrocytes
(Yao et al., 2005) and in a subpopulation of reactive astrocytes in human MS lesions (Geurts et al., 2005), and is relatively recently come to light as a potential therapeutic target in Parkinson’s disease (Amalric et al., 2013). Furthermore, while it is generally held that mGluR5 is downregulated in astrocytes and oligodendrocytes during development (Romano et al., 1996; Cai et al., 2000;
Romano et al., 2002; Luyt et al., 2006), its levels in the optic nerve were highest at 18-months, as were mGluR4 and mGluR7 compared to 1-month nerves, suggesting a role in the ageing process.
A key feature of glia throughout the CNS is their expression of functional
P2 receptors, and in the optic nerve the main transcripts were for the metabotropic P2Y12, P2Y6 and P2Y13 receptors and ionotropic P2X4 and P2X7 receptors, which are the substrate for glial Ca2+ signalling (James & Butt, 2002).
Glia can express all P2X subunits, but my results are consistent with P2X4 and
P2X7 subunits predominating in optic nerve astrocytes, oligodendrocytes and microglia (Agresti et al., 2005a; Agresti et al., 2005b; Matute et al., 2007b; Butt,
2011), and are of interest because they can cause sustained influx of Ca2+ and
117 mediate glial pathological responses, in particular the loss of oligodendrocytes and myelin in ischemia and demyelination (Matute & Cavaliere; Matute et al.,
2007b; Domercq et al., 2010). Notably, P2Y12 and P2Y13 receptors are prominent in microglia (McIlvain et al., 2010), but oligodendrocytes also express P2Y12 where they are believed to contribute to myelination (Amadio et al., 2006;
Amadio et al., 2010; Butt, 2011), and P2Y13 receptors in astrocytes mediate calcium signals (Carrasquero et al., 2009). The marked developmental downregulation of P2Y12R strongly suggests it is important for myelination, whereas the increase in P2Y13 may indicate changes in microglial and/or astrocyte function that are important in ageing white matter.
3.3.4 Axonal genes and synaptic release mechanisms in the optic nerve
A number of axonal transcripts were highly expressed, including neurofilament (Nefl), which is specific to axons, and ankyrin-3G (Ank3), which is an axolemmal protein that anchors sodium channels at nodes of Ranvier, although the key nodal sodium channel, Nav1.6 was called absent (Schafer et al.,
2006; Schafer & Rasband, 2006). These axonal transcripts were highest in the developing optic nerve, which is consistent with the reported prominence of local translation of mRNA by axonal ribosomes, and the apparent increase in Nefl transcripts in the ageing optic nerve may be a sign of axonal damage (Bomont et al., 2000). This microarray data requires further examination by qRT-PCR, but may be informative of the axonal transcriptome. For example, transcripts for the synaptic vesicle glycoprotein 2B (Sv2b) and Synaptotagmin-1 (Syt1) were
118 detected at relatively high levels in the postnatal nerve. Both Sv2b and Syt1 act via presynaptic calcium to regulate GABA and glutamate release regulate calcium is expressed at axonal terminals (Bragina et al., 2011), and their expression in the postnatal nerve is consistent with higher transport of their mRNA to the axon terminals during the postnatal period of active synapse formation. Syt1 has also been shown to regulate axon growth (Greif et al., 2013) and its apparent upregulation in the ageing nerve may indicate altered axonal growth or plasticity. In support of this possibility, a number of prominent transcripts that encode synaptic proteins were detected in the developing optic nerve. Notable amongst these were the high levels of transcripts for synaptobrevin-3 (Vamp3),
Vapa (Vesicle associated protein A), SNAP proteins (Napa), and syntaxin 7 (Stx7), which as part of the SNAP-SNARE complex play a critical role in vesicle trafficking, docking and fusion, and were highest in the postnatal nerve.
Furthermore, it is possible these mRNAs represent local translation of synaptic proteins, since axonal vesicular release mechanisms for glutamate have been described in the developing optic nerve (Alix et al., 2008). However, the main vesicular transporters for glutamate, GABA and ATP were called absent or barely detectable in the postnatal optic nerve, suggesting against local translation of axonal synaptic proteins.
119 3.3.5 Astrocytes as a source of synaptic neurotransmitter release in the optic
nerve
It is important to note that astrocytes are a potential source of neurotransmitters and are capable of vesicular neurotransmitter release (see
(Zorec et al., 2012) for recent review). Notably Adk (adenosine kinase) was prominent at all ages and encodes for an enzyme that catalyzes ATP to adenosine, both intracellularly and extracellularly, consistent with the prominent role for ATP- and adenosine-mediated signalling in the optic nerve (Butt, 2011).
In addition, a number of enzymes involved in the formation and breakdown of glutamine, GABA and glutamate were expressed at all ages. Notably, Gad1
(GAD67) and Gad2 (GAD65) are expressed by astrocytes and required for the synthesis of GABA from glutamate; both were upregulated in the ageing nerve.
An interesting feature was that the enzymes responsible for the endocannabinoid 2-arachidonoylglycerol (2-AG), monoglyceride lipase (Mg11) and diaglycerol lipase (Daglb), were differentially regulated with age: Mg11 was most highly expressed in the postnatal nerve and Daglb was increased in the mature and ageing nerve. 2-AG is produced by astrocytes, microglia and oligodendrocytes and, in addition to being anti-inflammatory, plays a critical role promoting OPC differentiation and myelin formation (Gomez et al., 2010).
Vesicular release in astrocytes depends on the core SNAP-SNARE complex, including synaptobrevin-2 (Vamp2), its homologue cellubrevin
(Vamp3), syntaxins and SNAP-23 (Verkhratsky and Butt, 2013). An analysis of
120 synaptic terminals in the hippocampus found astrocytes abundantly express all three protein partners necessary to form an operational SNARE complex, but not the same isoforms as neighbouring neuronal synaptic terminals (Schubert et al.,
2011): astrocytes expressed SNAP23 and VAMP3, whereas neurones expressed
SNAP25 and VAMP2, and syntaxin 1 was common to both. In the optic nerve,
SNAP23 and Vamp3 were most enriched in the postnatal nerve, and the main syntaxins were syntaxin 6 and 7. Astrocytes can also express SNAP25 and VAMP3
(Wilhelm et al., 2004; Crippa et al., 2006). Notably, in the ageing optic nerve there was a marked shift to SNAP25/VAMP3/syntaxin12, together with an upregulation of complexin-1 (Cplx1), which binds syntaxin and promotes vesicular release. This implies major changes in astrocyte and/or axonal synaptic mechanisms that may be important in the age-related decline in white matter function and require further investigation.
3.4 Summary and conclusions
The results of this section demonstrate that multiple neurotransmitter- signalling mechanisms are a prominent feature of the optic nerve, and provide a neurotransmitter receptor profile of optic nerve glia (Figure 3.15). There is pre- eminence of glutamate, ATP and GABA signalling, and an important finding is that they were expressed at all ages, since most studies have been on postnatal white matter and it was suggested neurotransmitters may have been a developmental phenomenon. A number of signalling mechanisms were upregulated in the ageing optic nerve, most notably GABA signalling, mGluR5
121 and P2Y13R, and the synaptic proteins VGluT1 and SNAP25. A key function for neurotransmitter signalling is in the regulation of oligodendrocyte differentiation, and changes in the 18-month optic nerve may be relevant to the observed decrease in OPC genes and the reported loss of white matter in the ageing brain. In respect to this, the ageing optic nerve was characterized by increased inflammation and deregulation of proteostasis, which may be important in age-related pathological changes in white matter.
122
Section 4
Functional expression of metabotropic
glutamate receptors in the
mouse optic nerve
123 4.1 Introduction and aims
Glutamate is an important neurotransmitter in the optic nerve, with considerable evidence that it acts on ionotropic glutamate receptors (iGluRs) in oligodendrocytes and astrocytes (Butt et al., 2014a). iGluRs are ligand-gated ion channels permeable to K+, Na+ and Ca2+ and are classified into three groups, namely NMDA (N-Methyl-D-Aspartate), AMPA (α-amino-3-hydroxyl-5-methyl-4- isoxazole-propionate), and Kainate receptors, with immunohistochemical, functional calcium imaging, RT-PCR and western blot evidence of all subtypes being expressed in the optic nerve (reviewed in (Butt et al., 2014a)). There is less evidence for mGluRs in the optic nerve, although they are reported to be widely expressed in glial cells in other areas of the brain (Verkhratskiĭ & Butt, 2013). mGluR are seven transmembrane domain G protein-coupled receptors (GPCR) and are divided in three groups, based on their second messenger coupling, pharmacology and amino acid sequence (Pin & Bockaert, 1995). Group I mGluRs
(mGluR1 and mGluR5) are positively coupled to phospholipase C (PLC) via Gq/11 and inositol triphosphate (IP3) formation, whilst group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, mGluR7 and mGluR8) are negatively coupled to adenylyl cyclase via Gi/G0. Each receptor subtype exhibits a well-defined expression pattern in several brain regions. In neurons, group I mGluRs are generally found in postsynaptic densities and modulate postsynaptic activity, whereas group II and III mGluRs are mainly postsynaptic and regulate neurotransmitter release (Ferraguti & Shigemoto, 2006). mGluRs are also expressed by glial cells, in which they modulate various glial functions and have a
124 role in glial-neuronal interaction. The microarray analysis in Section 3 identified
RNA transcripts for mGluRs in the mouse optic nerve, but their functional expression has not previously been determined. Therefore, the aim of this section is to examine the protein expression of mGluRs in the mouse optic nerve compared to the brain, using qRT-PCR and immunocyto- and immunohistochemistry, and to determine their functionality by performing calcium imaging on intact isolated mouse optic nerves.
125 4.2 Results
4.2.1 qRT-PCR analysis of mGluRs in mouse optic nerve and brain
Developmental changes in the mGluR expression was analysed in the cortex, cerebellum and optic nerve of mice aged P9 (pre-myelination), P12
(active myelination) and P30 (post myelination/mature), performing qRT-PCR, using a custom RT2 ProfilerqPCR array (Sabiosciences, Qiagen). The melt curve analysis shows that single amplicons are generated by qPCR (Figure 4.!) Results are expressed as relative gene expression using the 2^-Ct method, normalised to Gapdh. In the cortex, transcripts for mGluR3 (Grm3) and mGluR5 (Grm5) are the most abundant at P9 and are downregulated by P12 and remain at the same level at P30 (ANOVA and post hoc Bonferroni's tests, p values indicated on Figure
4.1A); the other mGluR are detected at low levels, with the exception of mGluR6, which was not detected (Figure 4.2A). In the cerebellum, mGluR4 (Grm4) is markedly upregulated between P9 and P12 and is the most highly expressed mGluR in the P30 cerebellum (Figure4.2B; p<0.001); the other mGluRs are present at low levels or absent. In the optic nerve, mGluR1 (Grm1), mGluR3
(Grm3), and mGluR5 (Grm5) are the most highly expressed and are developmentally upregulated (Figure 4.2C; p<0.001); mGluR4 (Grm4) and mGluR7 (Grm7) are also evident in P30 nerves, whereas the remaining mGluR are barely detectable or absent.
126
Figure 4.1 qRT-PCR melt curve analysis. Graph of the negative first derivative of representative melting-curves for all 8 mGluR subunits in a typical qRT-PCR experiment. Every trace represents a different mGluR. The presence of a single peak suggests that a single, specific product was amplified in the essay.
127
A
B
C
Figure 4.2 Expression of mGluR transcripts in cortex, cerebellum and optic nerve of P9, P12 and P30 mice. Tested for significance by ANOVA and post hoc Bonferroni's test (*p<0.05, **p<0.01, ***p<0.001).
128 4.2.2 Group I and II mGluRs are expressed in the grey and white matter of the
mouse brain
In light of the qRT-PCR analysis above and the known functions of mGluR in glia, I focused my immunohistochemical analysis on the group I subunit mGluR5 and group II subunit mGluR3; mGluR1 was also examined, but the antibodies were not successful. Because of their high sequence homology, the anti-mGluR3 antibody also recognizes mGluR2, but the qRT-PCR data indicates that immunostaining is likely to reflect mGluR3 rather than mGluR2. It is known that mGluR subtypes have a specific regional pattern (Ferraguti & Shigemoto,
2006) and my results in P8 coronal brain sections are consistent with the literature on immunostaining for mGluR5 and mGluR2/3 (Figure 4.3 and 4.4). An interesting observation is the difference in mGluR5 and mGluR2/3 immunostaining between grey and white matter: the receptors are in fact highly expressed in thalamic regions, whereas their expression in the neighbouring corpus callosum appears significantly low in comparison (Figure 4.3 A-Aii and
Figure 4.4 A-Aii). Because of the punctate nature of mGluR immunostaining, it is difficult to identify specific glial cell expression, although qualitative examination demonstrates the close association of mGluR with the EGFP+ astrocytes and
NG2+ cells, these could represent close juxtaposition to neuronal synaptic mGluR
(Figure 4.3 B-Cii and Figure 4.4 B-Cii). To address this, the Volocity 3D Image
Analysis Software (PerkinElmer) was used to create colocalisation channels to investigate whether mGluRs and EGFP or dsRed reporter are present in the same voxels, which provides evidence of colocalization of both mGluR5 and mGlur2/3
129 with astrocytes and NG2+ cells (Figure 4.5). There is evidence of localization of mGluR5 and mGluR2/3 to astrocyte cell bodies, processes and endfeet ensheathing blood vessels (Figure 4.5A-Bii). Similarly, mGluR5 and mGlur2/3 are localized on pericytes, NG2-DsRed+ cells that are juxtaposed to blood vessels
(Figure 4.5 C-Dii), as well as parenchymal NG2-DsRed+ cells with the morphology of OPCs (Figure 4.5 Di and Dii; data not shown for mGluR5).
130
Figure 4.3 mGluR5 expression in grey and white matter of the mouse forebrain. Confocal images of brain sections from P8 NG2-dsRed (A-Aii and C-Cii) and GFAP-EGFP mouse (B-Bii) immunolabelled for mGluR5. (A-Aii) Coronal brain section of a P8 NG2- dsRed reporter mouse showing higher mGluR5 expression level in the thalamus compared to the corpus callosum. (B-Bii) High magnification confocal images ofthe mGluR5 staining in the thalamus of a P8 GFAP-EGFP mouse. (C-Cii) High magnification confocal images of the mGluR5 staining in the thalamus of a P8 NG2-dsRed mouse. Individual channels and the overlay are illustrated. mGluR5 immunostaining was absent in negative controls (not illustrated). Scale Bars: 100µm in A-Aiii and 20µm in B-Cii.
131
Figure 4.4 mGluR2/3 expression in grey and white matter of the mouse forebrain. Confocal images of brain sections from P8 GFAP-EGFP (A-Bii) and NG2-dsRed mouse (C- Cii) immunolabelled for mGluR2/3. (A-Aii) Coronal brain section of a P8 GFAP-EGFP reporter mouse showing higher mGluR2/3 expression level in the thalamus compared to the corpus callosum. (B-Bii) High magnification confocal images of the mGluR5 staining in the thalamus of a P8 GFAP-EGFP mouse. Individual channels and the overlay are illustrated. (C-Cii) High magnification confocal images of the mGluR5 staining in the thalamus of a P8 NG2-dsRed mouse. mGluR2/3 immunostaining was absent in negative controls (not illustrated). Scale Bars: 100µm in A-Aiii and 20µm in B-Cii
132
Figure 4.5 mGluR5 and mGluR2/3 colocalisation with astrocytes, pericytes and NG2 cells in grey matter of the mouse forebrain. Confocal images of brain sections from P8 GFAP-EGFP (A-Bii) and NG2-dsRed mouse (C-Dii) immunolabelled for mGluR5 (A-Aii and C-Cii) and mGluR2/3 (B-Bii and D-Dii). (A-Aii) mGluR5 immunostaining showing the colocalisation with GFAP-EGFP (Ai, colocalised voxels appear yellow) and the colocalisation channel (Aii). (B-Bii) mGluR2/3 immunostaining showing colocalisation with GFAP-EGFP (Bi, colocalised voxels appear yellow) and the colocalisation channel (Bii). (C-Cii) mGluR5 immunostaining showing the colocalisation with NG2-DsRed (Ci, colocalisation appears yellow) and the colocalisation channel (Cii). (D-Dii) mGluR2/3 immunostaining showing colocalisation with NG2-dsRed (Di, colocalisation appears yellow) and the colocalisation channel (Dii). Scale Bars: 20µm.
3 13
4.2.3 Group I and II mGluR expression in mouse optic nerve glia
mGluR5 and mGluR2/3 immunolabelling was performed on 14µm optic nerve cryosections from P9 GFAP-EGFP mice. Compared to the widespread expression observed in the brain, mGluR immunolabelling was low in the optic nerve (Figure 4.6), and it was barely possible to detect colocalisation of mGluR5 or mGluR2/3 on glia, as illustrated in GFAP-EGFP+ astrocytes (Figure 4.6 Aii and
Bii), suggesting that mGluRs are more expressed in neurons than glial cells.
Although it was difficult to distinguish cellular expression in optic nerve sections, the qRT-PCR above indicated robust expression of mGluR5 and mGluR3 transcripts in the optic nerve. Hence, to confirm that optic nerve glia express mGluR5 and mGluR2/3, explant cultures were prepared from optic nerves from
P8 GFAP-EGFP and PLP1-dsRed reporter mice and examined after 5 days in vitro
(DIV) (Figure 4.7). The results demonstrate that optic nerve astrocytes identified by their expression of the GFAP-EGFP reporter express mGluR5 on their cell somata (Figure 4.7 A-Aii) and mGluR2/3 in their cell bodies and primary processes (Figure 4.7 C-Cii). Oligodendrocytes identified by their expression of the PLP1-dsRed reporter are shown to express mGluR5 and mGluR2/3 on their cell somata and processes (Figure 4.7 B-Bii and D-Dii).
134
Figure 4.6 mGluR5 and mGluR2/3 immunolabelling in the mouse optic nerve. Confocal images of optic nerve sections from P9 GFAP-EGFP mice immunolabelled for mGluR5 (A- Aii) and mGluR2/3 (B-Bii). (A-Aii) mGluR5 immunostaining showing colocalisation with GFAP-EGFP (Ai, colocalisation appears yellow), and the colocalisation channel (Aii). (B- Bii) mGluR2/3 immunostaining showing little colocalisation with GFAP-EGFP (Ai, colocalisation appears yellow), and the colocalisation channel (Bii). Scale Bars: 20µm.
135
Figure 4.7 mGluR5 and mGluR2/3 immunolabelling in optic nerve glial cultures. Optic nerve explant cultures from P8 mice were analysed for mGluR5 and mGluR2/3 immunocytochemistry after DIV5. (A-Aii) mGluR5 immunostaining is evident mainly on the cell somata of astrocytes identified by expression of the GFAP-EGFP reporter, whereas mGluR2/3 immunostaining is present on the astrocytic cell body and primary processes (C-Cii). Immunolabelling for mGluR5 (B-Bii) and mGluR2/3 (D-Dii) is present on cell bodies and processes of oligodendrocytes identified by expression of PLP1-dsRed reporter. Individual channels and the overlay are illustrated. Scale Bars: 20µm.
6 13
4.2.4 Calcium imaging of mGluR function in optic nerve glia
Stimulation of group I mGluR leads to the activation of phospholipase C
(PLC), resulting in the formation of diacylglycerol (DAG) and inositol-(1, 4, 5)- triphosphate (InsP3), which leads to the release of calcium from intracellular stores. We therefore examined whether group I mGluR mediate Ca2+ fluxes in optic nerve glia using Fluo-4 calcium imaging, as described previously (Hamilton et al., 2008; Hamilton et al., 2010). In brief, P7-P13 nerves were isolated intact and loaded with Fluo-4AM, a green-fluorescent Ca2+ dye that in its AM
(acetoxymethylester) form is membrane permeable and, once taken up by the cell, cytosolic esterases remove the ester group and the dye is rendered membrane impermeable and thus remains intracellular (Garaschuk et al., 2006).
Fluo-4-loaded nerves were placed in a perfusion chamber under a Zeiss LSM5
Pascal Axioskop 2 confocal microscope and nerves imaged using a 20x/0.50
WPh2 Achroplan water immersion lens. Fluo-4 was visualised by excitation at
488 nm and optical z-sections were obtained, typically 7-8 sections at 2–3 µm intervals. Changes in Fluo-4 fluorescence were measured using the Zeiss LSM
Image Examiner software (Zeiss, Germany) in glial cell bodies, selected as regions of interest (ROI). The vast majority of cells loaded in the optic nerve are considered to be astrocytes (Fern, 1998), although oligodendrocytes and OPCs may also take up the calcium indicator, and possibly the small population of microglia (Hamilton et al., 2010). ATP is known to mediate robust and
2+ reproducible [Ca ]i elevations in optic nerve glia and hence its application
(100μM) is used as positive control(Hamilton et al., 2008). On average, 11% of
137 the cells that responded to ATP were also responsive to the group I/II mGluR agonist (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid (ACPD).
Figure 4.8A illustrates paired recordings from P9 optic nerve glia showing the response to ATP, glutamate and ACPD (all at 100 M; n=41 cells from 5 nerves). The mean relative potency of action was ATP > glutamate > ACPD
(Figure 4.7C). However, the response to the agents differed between cells, for example some cells displayed an equal response to all three agents, and others a greater response to ACPD than ATP or glutamate (Figure 4.8B). In addition, the primary response to the agents was characterised by a single peak, but some cells exhibited multiple peaks in response to glutamate and ACPD (Figure 4.8B).
In an attempt to examine whether the variation in responses to ACPD might reflect different glial cells types, the diameter (d) of cell somata was measured and those with large somata (d11m) were considered putative astrocytes, and those with small somata (d10m) putative non-astrocytes, comprising mainly of oligodendrocytes, but also smaller populations of OPC and microglia. Interestingly, many of the smaller cells displayed the largest responses to ACPD, but did not respond to ATP (Figure 4.9, red traces), whereas most putative astrocytes had large responses to ATP and smaller response to ACPD.
These results illustrate the need for a method of identifying glial cells in these experiments, but in the absence of an unequivocal method this analysis was not
138 pursued further, and in subsequent experiments cells were selected on the basis of a positive response to ACPD.
Figure 4.8 Comparison of response to ATP, glutamate and ACPD in optic nerve glia. (A) Confocal images of Fluo-4 fluorescence intensity (illustrated in rainbow false colour) and (B) representative traces of individual glial cells illustrate the changes in fluorescence in response to ATP, glutamate and group I/II mGluR agonist ACPD (all at 100 M and administered for 30s). (C) Bar graph showing mean response (± SEM) expressed in arbitrary units (n=41 cells from 5 nerves). *p>0.5, ***p>0.001, paired t-tests. Scale bar 15 μm.
139
Figure 4.9 Calcium responses in optic nerve glia in response to the group I/II agonist ACPD. Raw traces of individual regions of interest (ROIs) illustrate the changes in Fluo-4 fluorescence in response to 4 administrations of 100 M ACPD (30s each, with 10min recovery in between), followed by 100 M ATP (30s). The diameter (d) of the cells was measured and cells were considered putative astrocytes (d11m; black traces) and putative non-astrocytes (oligodendrocytes, OPC, pericytes or microglial cells; d10m; red traces).
140 2+ 4.2.5 Group I mGluR mediated raised [Ca ]i in optic nerve glia
Paired recordings from Fluo-4 AM loaded cells show that cells responding to ACPD also responded to the specific group I agonist DHPG (100μM) (Figure
2+ 4.10A), and the mean rise in glial [Ca ]i was significantly greater for DHPG than
ACPD in paired recordings (Figure 4.9; n=13 cells from 4 nerves; p<0.01, paired t- test). However, the response varied between cells, with ACPD evoking a larger response in some cells and DHPG in others (Figure 4.10B). In addition, the response to ACPD was generally a single or double peak, whereas DHPG evoked multiple peaks, characteristic of astroglial Ca2+ oscillations (Figure 4.10B).
The group I mGluR antagonist AIDA (100μM) significantly inhibited the glial response to ACPD (Figure 4.11; n=16 cells from 4 nerves, p<0.001, paired t- test), Interestingly, the blockade of group I mGluR alters the kinetics of the ACPD response, which in the presence of AIDA was characterised by multiple peaks or oscillations (Figure 4.11B). ACPD activates both group I and group II mGluR, whereas AIDA specifically blocks group I mGluR, and the variable effect of AIDA between cells identified cells in which AIDA completely blocked the response to
ACPD in some cells (Figure 4.11A, ‘group I responder’), but had no effect in other cells (Figure 4.10A, ‘group II responder’). Although group II mGluR are coupled to cAMP and not Ca2+, these results raise the possibility that group II mGluR may be involved in glial calcium signaling. However, it was not possible to follow up these observations, due to malfunction of the calcium imaging equipment.
141
Figure 4.10 Group I mGluR mediate calcium signals in optic nerve glia. (A) Confocal images of Fluo-4 fluorescence intensity (illustrated in rainbow false colour) and (B) representative traces of individual glial cells illustrate the changes in fluorescence in response to ACPD and selective group I mGluR agonist DHPG (both at 100 M, applied for 30s). (C) Bar graph of mean response (± SEM) expressed in arbitrary units (n=13 cells from 4 nerves). **p<0.01, paired t-test. Scale bar 10μm.
142
Figure 4.11 Group I mGluR blockade significantly decreases the response to ACPD. (A) Confocal images of Fluo-4 fluorescence intensity (illustrated in rainbow false colour) and (B) representative traces of individual glial cells illustrate the changes in fluorescence in response to ACPD in the aCSF and in the presence of the specific group I mGluR agonist AIDA (both at 100 M). (C) Bar graph shows mean response (± SEM) expressed in arbitrary units (n=16 cells from 4 nerves). ***p<0.001, paired t-test. Scale bar 10μm.
143 4.3 Discussion
The results of this section show that optic nerve glial cells express group I and group II mGluRs at mRNA and protein level, and the calcium imaging analysis demonstrated that these receptors are functional.
4.3.1 qRT-PCR limitations
In this study, gene expression was determined by the Relative
Quantification method, where the quantity of genes of interest is expressed relative to reference genes within the same sample. The optimal way to determine the most appropriate reference genes with the lowest difference between samples would be to use RNA sequencing or to run a variety of candidate reference genes in triplicates for all samples and different experimental conditions. These methods were not feasible in my study because of the large number of mice that would require. The appropriate reference gene
(Gapdh) was selected by the standard deviation method, in combination with the
NormFinder algorithm (Casadei et al., 2011). Gapdh is the most widely used reference gene across the literature and has been shown to be the most stable gene in the mouse brain during development and in ageing (Sieber et al., 2010;
Kraemer et al., 2012).
The developmental analysis of the expression of mGluR has been performed on extracts of pooled whole mouse optic nerves, therefore the
144 expression profile of a specific cell type could not be determined. Nonetheless, the vast bulk (>90% of cells) of the RNA extracted in whole optic nerves is from astrocytes and oligodendrocytes (Salter & Fern, 2005). Hence, it can be hypothesised that the most highly expressed subunits identified in my study, such as mGluR1, mGluR3 and mGluR5 are in astrocytes and/or oligodendrocytes.
This is in partial agreement with published studies that detected mRNA for mGluR3 and mGluR5, but not mGluR1, in hippocampal astrocytes isolated from young and adult rats (Schools & Kimelberg, 1999; Cai et al., 2000) and OPCs (Luyt et al., 2003). MGluR1 has been detected by immunocytochemistry in 10% of cultured astrocytes prepared from spinal cord (Silva et al., 1999) and in a subpopulation of reactive astrocytes in multiple sclerosis lesions (Geurts et al.,
2003). The expression of mGluR5 in astrocytes and mGluR3 and mGluR5 in oligodendrocyte lineage cells decreases during development (Cai et al., 2000;
Luyt et al., 2006), similarly to that reported in neurons (Romano et al., 1996;
Romano et al., 2002). This profile might be masked or distorted in our study because of the heterogeneity of the tissue and the concomitant presence of cells at different stages of maturation. With the exception of mGluR6, whose expression is restricted to retinal bipolar cells, all mGluR subunits are expressed in the mouse cortex, with mGluR3 and mGluR5 at the highest level. On the other hand, the most highly expressed in the cerebellum was mGluR4 (GRM4), which is expressed by granule cell axons, but also other subunits (mGluR1, mGluR2, mGluR5, mGluR7) are expressed in the cerebellum (Knöpfel & Grandes, 2002). It is likely that the most expressed transcripts in the optic nerve are the most important functional receptors, but it is important to take into consideration that
145 levels of mRNA do not directly correlate to the protein expression level. In fact, there are many different post-transcriptional and post-translational regulatory mechanisms that may lead to mismatches between mRNA and protein levels
(Greenbaum et al., 2003; Vogel & Marcotte, 2012).
4.3.2 mGluR5 and mGluR2/3 expression in the mouse optic nerve
Our immunohistochemical analysis of mGluR5 and mGluR2/3 first of all showed a dramatic difference in expression levels between grey matter and white matter, suggesting that these receptors are expressed mainly by neurons.
Nonetheless, it is known that astrocytes express mGluR5 and that its activation leads to an increase in intracellular calcium levels. This, in turn, results in the release of glutamate and other gliotransmitters, which can evoke synaptic responses in neighbouring neurons and glia (Pasti et al., 1997; Bezzi et al., 1998).
This mechanism is the reason why mGluR5 is involved in a plethora or functions, such as learning and memory, synaptic plasticity, ion channel regulation, cellular excitability, anxiety and perception of pain. As a consequence, dysregulation or misexpression of mGluR5 is associated with major diseases, e.g. anxiety, fragile-X syndrome, epilepsy and AD (Niswender & Conn, 2010). Consistent with my qRT-
PCR findings, astrocytes have been shown to express mGluR3 but not mGluR2 in vivo (Petralia et al., 1996; Ohishi et al., 1998; Tamaru et al., 2001; Mudo et al.,
2007) and in vitro (Ciccarelli et al., 1997; Aronica et al., 2003). In addition, mGluR2/3 immunoreactivity has been found in microglia/macrophages in biopsies from patients with multiple sclerosis (Geurts et al., 2003). The activation
146 of group II mGluRs in cultured astrocytes can have a dual regulation: it reduces the forskolin-stimulated cAMP formation in the absence of extracellular calcium, but enhances it in the presence of calcium (Moldrich et al., 2002). Moreover, group II mGluR stimulation amplifies the stimulatory effect on cAMP formation mediated by 2-adrenergic receptors, and this combined activation stimulates the release of adenosine in cultured astrocytes (Winder et al., 1996). Adenosine is involved in the regulation of many glial functions, such as glial proliferation, glutamate transporters expression and glutamate sensitivity (Daré et al., 2007;
Verkhratsky et al., 2009; Boison et al., 2010).
Although is not clear whether mGluR5 is expressed by oligodendrocytes in vivo, my results demonstrate optic nerve oligodendrocytes express mGluR5 in explants cultures, and cultured oligodendrocyes from other brain regions have been shown to express mGluR1, mGluR2/3, mGluR4 and mGluR5 (Deng et al.,
2003). The expression of mGluR is developmentally downregulated in culture, being high in early and late OPCs and low or absent in immature and mature oligodendrocytes(Deng et al., 2003). This suggest that mGluR may have a role in oligodendrocyte differentiation and myelination (Luyt et al., 2003). mGluR3 and mGluR5 have also been detected in OPCs from adult human brain (Luyt et al.,
2003; Luyt et al., 2004). The activation of mGluR5 protects OPCs from staurosporine-induced apoptosis (Luyt et al., 2006), suggesting that its downregulation in premyelinating OLs increases their vulnerability. Moreover,
2+ consistent with my study showing that ACPD evokes raised [Ca ]i in optic nerve
147 glia, the administration of ACPD has been shown to attenuate the loss of white matter myelin in P6 rats subjected to hypoxia-ischemia (Jantzie et al., 2010).
4.3.3 mGluR at the gliovascular interface
My results draw attention to the similar pattern of expression of mGluR3 and mGluR5 at the interface between astrocytes and pericytes, both members of the neurovascular unit. The neurovascular unit is the point of contact on a blood vessel of astrocytes, endothelial cells, and pericytes. It plays a crucial role in neurovascular coupling. Neurovascular coupling, also referred to as functional hyperaemia, is the phenomenon by which an intensification of neuronal activity leads to a local increase in blood flow. When a rise in neural activity occurs, neurotransmitters are released and the metabolic demand rises, with an increase of O2, glucose and ATP consumption. Cerebral blood flow (CBF) increases to provide a continuous supply of nutrients and supports the normal functioning of the brain (Ventura & Harris, 1999; Simard et al., 2003). The functional relevance of this connection has been the subject of several studies.
Synaptically released glutamate causes astrocytes to produce and release many vasoactive compounds (Petzold & Murthy, 2011), such as ATP (Queiroz et al.,
1999; Arcuino et al., 2002; Coco et al., 2003), nitric oxide (NO) (Murphy et al.,
1993; Li et al., 2003), arachidonic acid (AA) metabolic products, such as prostaglandin E2 (PGE2), and epoxyeicosotrienoic acids (EETs) (Pearce et al.,
1989; Amruthesh et al., 1993; Roman, 2002). These substances are believed to act on smooth muscle cells in arterioles or on pericytes to determine the vascular
148 tone, contraction or dilation, depending on the local O2 concentration (Zonta et al., 2003b; Gordon et al., 2008; Attwell et al., 2010). Specifically, the activation of the astrocytic mGluR5 leads to the pulsatile release of PGE2, which causes dilation of arterioles (Zonta et al., 2003a; Zonta et al., 2003b; Filosa et al., 2004).
4.3.4 mGluR mediate calcium signals in the postnatal mouse optic nerve glia
Calcium is a key second messenger in glial physiology and pathology, as astrocytes use intercellular Ca2+ waves as a mechanism for communication
(Verkhratsky et al., 1998). The combined expression of the mRNA transcripts and immunostaining coupled with the response to ACPD and DHPG provide strong evidence for functional group I mGluR5 in optic nerve glia, in particular astrocytes. However, a major limitation of the calcium imaging experiments in the isolated intact optic nerve is that it was not possible to identify the specific glial cell types from which recordings were made, and experiments in the transgenic glial reporter mice was not successful. The vast majority of cells composing the optic nerve are astrocytes and oligodendrocytes(Salter & Fern,
2005), therefore we can assume that calcium imaging measurements will include both cell types. However, in my analysis only 11% of cells that responded to ATP also responded to the group I/II mGluR agonist ACPD. This indicates that most optic nerve astrocytes and oligodendrocytes do not express sufficient levels of mGluR5 to evoke a measurable calcium response, which is consistent with the immunohistochemical findings. The immunohistochemistry did not support the possibility that mGluR5 is expressed only by a small proportion of cells, and the
149 in vitro data demonstrated both astrocytes and oligodendrocytes express mGluR5.
The application of the group I/II mGluR ACPD agonist and group I specific
2+ agonist DHPG elicited arise in glial [Ca ]i, consistent with activation of mGluR5.
Surprisingly, the group I mGluR inhibitor AIDA did not completely abolish the
2+ increase in [Ca ]i evoked by ACPD, but changed its kinetics. In expression systems mGluR5 elicits oscillations, whereas the closely related mGluR1 evokes single-peaked responses (Kawabata et al., 1996; Nakanishi et al., 1998; Kettunen et al., 2002). In contrast, an activation of native mGluR5 in neurons can induce different effects: it elicits a single-peaked response in hippocampus (Rae et al.,
2000) and it induces oscillations in the neocortex (Flint et al., 1999). Astrocytes can display various pattern of response, transient or oscillatory, depending on the species, brain regions and age of the animal (McCarthy & Salm, 1991; Müller et al., 1997; Cai et al., 2000). In cultured astrocytes, which like optic nerve glia do not express mGluR1, the activation of mGluR5 with ACPD mediates calcium oscillations via PKC phosphorylation (Nakahara et al., 1997). In our experiments,
ACPD elicits mainly single-peaked responses, whereas DHPG, selective agonist for mGluR5, evokes an oscillatory response. ACPD does not evoke currents in white matter oligodendrocytes in brain slices (Káradóttir et al., 2005) although it
2+ elicits a rise in [Ca ]I in cultured OPCs (Holtzclaw et al., 1995). The expression of mGluR5 appears to decrease during development (Cai et al., 2000), and because of this DHPG induces calcium oscillations in the majority of OPCs, requiring Ca2+
150 release from intracellular stores, but only in a smaller proportion of oligodendrocytes (Luyt et al., 2003; Luyt et al., 2006). In OPCs, glutamate induced calcium oscillations could have a role in migration (Gudz et al., 2006). Although my calcium experiments were performed on P7-P13 optic nerves for technical reasons, the qRT-PCR did not support a possible down-regulation of mGluR5 with development in the optic nerve.
4.4 Summary and conclusions
The results of this section provide evidence of functional expression of group I mGluR5 and group II mGluR3 in optic nerve astrocytes and
2+ oligodendrocytes. Activation of mGluR5 evokes raised [Ca ]I in optic nerve glia, and there is evidence that group II mGluR may modulate these calcium responses. The expression of mGluR in the optic nerve is consistent with evidence that glutamate is an important signaling molecule and implies it has a physiological function; in astrocytes mGluR are implicated in their homeostatic functions and in oligodendrocytes in regulating differentiation and myelination.
The source of endogenous glutamate seems likely to be axons, which release glutamate in response to action potentials (Kukley et al., 2007; Ziskin et al.,
2007). Extensive studies on optic nerve anatomy have shown that astrocytes, oligodendrocytes and OPCs make contacts with axons at nodes of Ranvier (Butt et al., 1994a; Butt et al., 1994b; Butt et al., 1999) and glutamate and axonal
2+ activity can trigger [Ca ]i increase in optic nerve glia (Kriegler & Chiu, 1993;
151 James & Butt, 2001; Butt et al., 2004; Hamilton et al., 2008; Hamilton et al.,
2010). The results of this section show that mGluR are also involved in these signaling cascades.
152
Section 5
A protective role for glial metabotropic
glutamate receptors in ischaemia
153 5.1 Introduction and aims
In Section 4, I demonstrated that optic nerve astrocytes and oligodendrocytes express functional mGluR. The precise functions of mGluR in glia are unclear, although they are implicated in oligodendrocyte differentiation and in the tripartite synapse in grey matter astrocytes (Ferraguti & Shigemoto,
2006). In addition, like neurons, oligodendrocytes are highly susceptible to hypoxic/ischemic damage (Fern & Möller, 2000), and there is evidence that the activation of group I mGluRs might have a protective role in both neurons and oligodendrocytes exposed to ischaemic injury (Deng et al., 2003; Baskys et al.,
2005; Jantzie et al., 2010). Whilst in vitro studies have shown that cultured astrocytes are resilient to ischaemic injury (Goldberg & Choi, 1993), astrocytes in situ appear to be only slightly more resistant than neurons, and immature astrocytes may be more susceptible than adult (Shannon et al., 2007).
Furthermore, OPCs in developing white matter have inadequate anti-oxidative defences, which make them particularly sensitive to ischaemia (Fern & Möller,
2000; Back et al., 2001). Notably, ischaemic injury of developing white matter, termed periventricular leukomalacia (PVL) or periventricular white matter injury
(PWMI), is characterized by early damage of oligodendrocytes and axons and is the major cause of death and neurological impairment in cerebral palsy (Banker
& Larroche, 1962; Paneth, 1994; Volpe, 1995). In addition to oxygen and glucose deprivation, ischaemia is characterized by glutamatergic excitotoxicity caused by an increase in extracellular glutamate and the resulting over-activation of AMPA-
, KA- and NMDA-type glutamate receptors (Deng et al., 2003; Salter & Fern,
154 2005; Manning et al., 2008). In contrast, activation of mGluR in the face of raised extracellular glutamate is proposed to have a protective role in ischemia. The aim of this section is to examine the potential protective role of mGluR in astrocytes and oligodendrocytes in white matter ischemia, using the postnatal optic nerve and oxygen-glucose deprivation (OGD) model of ischemia/hypoxia
(Fern, 1998; Shannon et al., 2007; Salter & Fern, 2008).
155 5.2 Results
5.2.1 Group I/II mGluR activation protects optic nerve cells from ischaemia
In order to investigate the role of mGluR in glial cells during ischaemia, optic nerves were subjected to OGD (Salter & Fern, 2005; Hawkins & Butt, 2013).
Briefly, P8-P13 C57BL/6 wild-type (wt) mice and GFAP-EGFP, SOX10-EGFP, PLP1-
DsRed, NG2-DsRed reporter mice were killed humanely and the optic nerves rapidly isolated intact and immediately placed in aCSF (containing glucose – see section 2.5.4) in an incubator to stabilise for 30 min at 37C in normoxic conditions (95%O2/5% CO2). Optic nerves were then incubated for 1h at 37C in either normoxic (95%O2/5%CO2) and glucose aCSF, or in ZERO-O2 (95%N/5%CO2) and ZERO-glucose aCSF (OGD). All drugs were added directly to aCSF during incubation. At the end of the incubation, optic nerves were fixed in 4% PFA for
1h at RT, washed and mounted on microscope slides. Surviving cells expressing
EGFP and DsRed reporters were visualised at 488nm and 568nm, respectively, using a LSM 710 Zeiss confocal microscope with a x40 oil immersion lens with high numerical aperture (1.3nm); images were captured maintaining the acquisition parameters constant between samples. Two images were captured at the mid-point along the length of each nerve, and 15 x 1 m z-sections were acquired, and the number of cells expressing the reporter was counted within a constant 20 x 20 m field of view (FOV) in the x-y-plane. Cell counts are expressed as mean SEM, where each ‘n’ value represents the number of optic nerves, and significance was determined by ANOVA and post-hoc Newman-Keuls multiple comparison analysis using Prism5.0 (Graphpad) (Hawkins & Butt, 2013). 156 Firstly, to determine whether mGluR are cytoprotective in OGD, optic nerves from P10 C57BL/6 wild-type mice were subjected to OGD in the presence of the general Group I/II mGluR agonist 1-Amino-1,3- dicarboxycyclopentane (ACPD, 100 M) and propidium iodide (PI) was used to identify dead cells. Compared to normoxic control nerves incubated in
O2+glucose (Figure 5.1A), OGD resulted in a statistically significant increase in the number of PI+ cells (Figure 5.1C, E; p<0.05), demonstrating that OGD induced cell death. Incubation with ACPD in OGD significantly decreased the number of PI+ cells compared to OGD in aCSF (Figure 5.1D, E; p<0.01), to a level observed in normoxic control conditions. Incubation with ACPD in normoxic conditions did not have a significant effect on the number of PI-positive cells (Figure 5.1B, E).
157
Figure 5.1 Effect of the Group I/II mGluR agonist ACPD on OGD induced cell death in postnatal mouse optic nerve. Optic nerves from P10 C57BL/6 mice were exposed to 1 h OGD in the absence or presence of the Group I/II mGluR agonist ACPD (100 µM) and compared to normoxic controls incubated in O2+glucose (O2). (A–D) Representative + images of PI cells in control O2 (A), O2+ACPD (B), OGD (C), and OGD+ACPD (D); scale bars = 50 m. (E) Quantification of the number of PI+ cells in constant fields of view
(FOV) (MEAN ± SEM, n ≥ 5 nerves per experimental group; *p<0.05, **p<0.01, ANOVA with Newman–Keuls multiple comparison post-hoc analysis).
158 5.2.2 Astrocytes in the postnatal optic nerve are susceptible to OGD and are
protected by mGluR activation
In order to understand which cells are protected by mGluR activation and examine the mGluR subtypes involved, the same experimental protocol as above was performed using postnatal nerves from different reporter mice to identify the different glial cell types, and the group I mGluR agonist DHPG ((S)-3,5-
Dihydroxyphenylglycine) and group II mGluR agonist LY379268. Analysis of nerves from GFAP-EGFP mice demonstrates that astrocytes are susceptible to
OGD (Figure 5.2B), with a marked 50% decrease in GFAP-EGFP+ cells in OGD compared to normoxic controls (Figure 5.2A, F, p<0.001). Incubation with ACPD,
DHPG, or LY379268 all had an equivalent protective effect against OGD in astrocytes (Figure 5.2C-F), rescuing astrocyte numbers to that seen in controls
(see Figure 5.2F for details of p values).
159 Figure 5.2 mGluR activation protects postnatal optic nerve astrocytes from OGD. Optic nerves from P9 GFAP-EGFP reporter mice were exposed to 1 h OGD in the presence or absence of the group I/II agonist ACPD, or specific group I agonist DHPG and group II agonist LY379268 (all 100 µM), compared to control nerves incubated in normal aCSF
+ with O2+glucose (O2). (A–E) Representative images of GFAP-EGFP astrocytes in isolated intact optic nerves incubated in O2 (A), OGD (B), OGD+ACPD (C), OGD+DHPG (D), or OGD+LY (E); scale bars = 50 m. (F) Quantification of the number of GFAP-EGFP+ cells in constant fields of view (FOV) (MEAN ± SEM, n ≥ 5 nerves per experimental group; ***p<0.001, ANOVA with Newman–Keuls multiple comparison post-hoc analysis).
160 5.2.3 Oligodendrocytes in the postnatal optic nerve are susceptible to OGD
and are protected by mGluR activation
To analyse oligodendrocyte lineage cells, I used optic nerves from P8
SOX10-EGFP mice (Figure 5.3) and P11 PLP1-DsRed mice (Figure 5.4). The Sox10-
EGFP reporter identifies both oligodendrocytes and OPCs, which are defined by their specific expression of the transcription factor Sox10, whilst the PLP1-DsRed reporter identifies mature oligodendrocytes and not OPC. Hence, any difference between SOX10-EGFP and PLP1-DsRed nerves is a measure of the changes in
OPCs. Compared to control normoxic nerves (Figure 5.3A), OGD resulted in a marked loss of SOX10-EGFP+ cells (Figure 5.3B), which was statistically significant
(Figure 5.3F, p<0.001). Incubation with ACPD, DHPG or LY379268 had a protective effect against OGD induced loss of Sox10-EGFP+ cells (Figure 5.3C-F; p<0.001); there was no significant difference between the different mGluR agonists, but the number of Sox10-EGFP+ oligodendrocytes in OGD+LY3799268 was significantly less than in control normoxic conditions (Figure 5.3F; p<0.01).
Similarly, OGD resulted in a significant loss of PLP-DsRed+ oligodendrocytes compared to normoxic controls (Figure 5.4A, B; p<0.001), and the mGluR agonists significantly protected against this (Figure 5.4C-F; p<0.001); mGluR agonists did not result in full recovery of PLP-DsRed+ cell numbers, which were significantly less than in normoxic numbers (Figure 5.4F; p<0.001).
161
Figure 5.3 mGluR activation protects postnatal optic nerve oligodendrocytes from OGD. Optic nerves from P8 SOX10-EGFP reporter mice were exposed to 1 h OGD in the presence or absence of the group I/II agonist ACPD, or specific group I agonist DHPG and group II agonist LY379268 (all 100 µM), compared to control nerves incubated in normal
+ aCSF with O2+glucose (O2). (A–E) Representative images of SOX10-EGFP oligodendrocyte lineage cells (Sox10-EGFP identifies all OPCs and oligodendrocytes) in isolated intact optic nerves incubated in O2 (A), OGD (B), OGD+ACPD (C), OGD+DHPG (D), or OGD+LY (E); scale bars = 50 m. (F) Quantification of the number of SOX10-EGFP+ cells in constant fields of view (FOV) (MEAN ± SEM, n ≥ 5 nerves per experimental group; **p<0.01, ***p<0.001, ANOVA with Newman–Keuls multiple comparison post-hoc analysis).
162
Figure 5.4 mGluR activation protects postnatal optic nerve mature oligodendrocytes from OGD. Optic nerves from P11 PLP1-DsRed reporter mice were exposed to 1 h OGD in the presence or absence of the group I/II agonist ACPD, or specific group I agonist DHPG and group II agonist LY379268 (all 100 µM), compared to control nerves incubated in normal aCSF with O2+glucose (O2). (A–E) Representative images of PLP1- DsRed+ oligodendrocytes (PLP1-DsRed identifies mature oligodendrocytes) in isolated intact optic nerves incubated in O2 (A), OGD (B), OGD+ACPD (C), OGD+DHPG (D), or OGD+LY (E); scale bars = 50 m. (F) Quantification of the number of PLP1-DsRed + cells in constant fields of view (FOV) (MEAN ± SEM, n ≥ 5 nerves per experimental group; ***p<0.001, ANOVA with Newman–Keuls multiple comparison post-hoc analysis).
163 5.2.4 Oligodendrocytes in the adult optic nerve are susceptible to OGD and are protected by mGluR activation
In order to understand if the mGluR mediated protective effect is specific to developing white matter undergoing myelination, OGD experiments were performed on optic nerves from adult (P33) PLP1-DsRed reporter mice. After 1h
OGD, there was a 30% loss of PLP1-DsRed+ oligodendrocytes compared to normoxic controls (Figure 5.5A, p<0.001). Incubation with the group I mGluR agonist DHPG protects oligodendrocytes from this OGD-induced loss (Figure
5.5C, p<0.05), whilst the group II mGluR agonist LY379268 was not significantly protective against OGD (Figure 5.5D, p>0.05). This result demonstrates that oligodendrocytes are susceptible to hypoxia/ischaemia in the adult optic nerve, and that mGluR are protective, but the results indicate this may be preferentially via group I mGluR.
164
Figure 5.5 mGluR activation protects adult optic nerve oligodendrocytes from OGD. Optic nerves from P33 PLP1-DsRed reporter mice were exposed to 1 h OGD in the presence or absence of the specific group I agonist DHPG or group II agonist LY379268 (both at 100 µM), compared to control nerves incubated in normal aCSF with
+ O2+glucose (O2). (A–D) Representative images of PLP1-DsRed oligodendrocytes in isolated intact optic nerves incubated in O2 (A), OGD (B), OGD+DHPG (C), or OGD+LY (D); scale bars = 50 m. (E) Quantification of the number of PLP1-DsRed + cells in constant fields of view (FOV) (mean ± SEM, n ≥ 5 nerves per experimental group; *p<0.05, **p<0.01, ***p<0.001, ANOVA with Newman–Keuls multiple comparison post-hoc analysis).
165 5.2.5 The protective effect of goup I mGluR on postnatal optic nerve
oligodendrocytes in OGD requires PKC activation
The results above indicate a cytoprotective role for group I mGluR, which are G-protein coupled receptors that act by an increase in phospholipase C (PLC) activity and subsequent formation of inositol triphosphate (IP3), which causes an increase of intracellular Ca2+ concentration and diacylglycerol (DAG), responsible for the activation of protein kinase C (PKC). In order to examine the pathways responsible for the group I mGluR mediated protective effect on oligodendrocytes, the PKC inhibitor Go6976 (10 M) was used in conjunction with DHPG, using the OGD model in optic nerves from P11 PLP1-DsRed reporter mice. The results demonstrate that the protective effect of DHPG is completely abolished by the inhibition of PKC (Figure 5.6, p<0.001), providing strong evidence that group I mGluR mediate their pro-survival effect on oligodendrocytes via PKC activation.
166
Figure 5.6 Protective effect of group I mGluR on postnatal optic nerve oligodendrocytes in OGD requires PKC activation. Optic nerves from P11 PLP1-DsRed reporter mice were exposed to 1 h OGD in the presence of the group I mGluR agonist
compared to normoxic controls incubated in normal aCSF with O2+glucose (O2). (A-D) Representative images of PLP1-DsRed+ oligodendrocytes in isolated intact optic nerves incubated in O2 (A), OGD (B), OGD+DHPG (C), or OGD+DHPG+Go6976 (D); scale bars = 50 m. (E) Quantification of the number of PLP1-DsRed + cells in constant fields of view
(FOV) (MEAN ± SEM, n ≥ 5 nerves per experimental group; *p<0.05, **p<0.01, ***p<0.001, ANOVA with Newman–Keuls multiple comparison post-hoc analysis).
167 5.2.6 Gq coupled receptors are protective against OGD in postnatal optic
nerve oligodendrocytes
Group I mGluR are coupled to Gq protein (Gq/11), a heterotrimeric G protein subunit that activates PLC. Many G-protein coupled receptors act via Gq proteins, including P2Y1 receptors that are expressed by oligodendrocytes (Butt,
2011). Therefore, to determine whether the cytoprotective effect of group I mGluR activation is specific to these receptors or possibly a general effect of all
Gq-coupled receptors, I examined the effects of the P2Y1 receptor specific agonist (N)-methanocarba-2MeSADP (MRS2365, 10 M) (Bourdon et al., 2006).
In P11 PLP1-DsRed optic nerves, activation of P2Y1 has a significant protective effect on oligodendrocytes in OGD (Figure 5.7B, D; p<0.05). The specificity of this effect was confirmed using the selective P2Y1 receptor antagonist 2-chloro N(6)- methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate (MRS2279, 10
M), which completely abolished the cytoprotective effect of MRS2365 (Figure
5.7C, D, p<0.01). This result raises the likelihood that Gq-coupled receptors have a general cytoprotective role against hypoxic/ischaemic injury in oligodendrocytes.
168
Figure 5.7 Activation of the Gq-coupled P2Y1 receptor protects postnatal optic nerve oligodendrocytes from OGD. Optic nerves from P11 PLP1-DsRed reporter mice were exposed to 1 h OGD in the presence or absence of the specific P2Y1 agonist MRS2365 and/or antagonist MRS2279 (both at 10 µM). (A-C) Representative images of PLP1- DsRed+ oligodendrocytes in isolated intact optic nerves incubated in OGD (A), OGD+MRS2365 (B), or OGD+MRS2365+MRS2279 (C); scale bars = 50 m. (D)
+ Quantification of the number of PLP1-DsRed cells in constant fields of view (FOV) (MEAN ± SEM, n ≥ 5 nerves per experimental group; *p<0.05, **p<0.01, ANOVA with Newman– Keuls multiple comparison post-hoc analysis).
169 5.2.7 Role of adenylate cyclase in the protective effect of group II mGluR on
postnatal optic nerve oligodendrocytes in OGD
The results presented above demonstrated that group II mGluR activation is protective for oligodendrocytes against OGD in postnatal nerves. Activation of group II mGluR leads to a decrease in adenylate cyclase (AC) and consequent decrease in cytosolic cAMP. With the aim of understanding the role of AC and cAMP in the group II mediated pro-survival effect on oligodendrocytes during
OGD, P11 PLP1-DsRed optic nerves were incubated with the lipid soluble cAMP analogue dibutyryl cAMP (dbcAMP, 100 M) (Figure 5.8), and the AC activator forskolin (100 M) (Figure 5.9), both of which act to raise cytosolic cAMP.
Incubation in dbcAMP completely blocked the protective effect of the group II mGluR agonist LY379268 on oligodendrocytes in OGD (Figure 5.8C, E, F, p<0.001); dbcAMP alone did not alter the effects of OGD (Figure 5.8D, F).
Similarly, incubation in forskolin, to activate AC and raise endogenous cAMP, significantly decreased the protective effect of LY379268 in OGD (Figure 5.9C, E,
F, p<0.05), and had no effect on OGD-mediated loss of oligodendrocytes (Figure
5.9B, D, F). The results provide evidence that the protective effect of group II mGluR is mediated by a decrease in cAMP, which can be reversed by pharmacologically raising cytoplasmic cAMP.
170
Figure 5.8 dbcAMP blocks the protective effect of group II mGluR on postnatal optic nerve oligodendrocytes in OGD. Optic nerves from P11 PLP1-DsRed reporter mice were exposed to 1 h OGD in the presence or absence of the group II mGluR agonist LY379268 (100 M), together with the cAMP analogue dbcAMP (100 M). (A-E) Representative
+ images of PLP1-DsRed oligodendrocytes in isolated intact optic nerves incubated in O2 (A), OGD (B), OGD+LY (C), OGD+dbcAMP (D), or OGD+LY+dbcAMP (E); scale bars = 50 m. (F) Quantification of the number of PLP1-DsRed + cells in constant fields of view
(FOV) (MEAN ± SEM, n ≥ 5 nerves per experimental group; **p<0.01, ***p<0.001, ANOVA with Newman–Keuls multiple comparison post-hoc analysis).
171
Figure 5.9 Forskolin blocks the protective effect of group II mGluR on postnatal optic nerve oligodendrocytes in OGD. Optic nerves from P11 PLP1-DsRed reporter mice were exposed to 1 h OGD in the presence or absence of the group II mGluR agonist LY379268 (100 M), together with the AC activator forskolin (100 M). (A-E) Representative
+ images of PLP1-DsRed oligodendrocytes in isolated intact optic nerves incubated in O2 (A), OGD (B), OGD+LY (C), OGD+forskolin (D), or OGD+LY+forskolin (E); scale bars = 50 m. (F) Quantification of the number of PLP1-DsRed + cells in constant fields of view
(FOV) (MEAN ± SEM, n ≥ 5 nerves per experimental group; *p<0.05, **p<0.01, ***p<0.001, ANOVA with Newman–Keuls multiple comparison post-hoc analysis).
172 5.3 Discussion
In this chapter, I have demonstrated that the activation of group I and II mGluR is cytoprotective for optic nerve astrocytes and oligodendrocytes in hypoxia/ischaemia in situ. Ischaemic cell death is characterised by an initial loss of energy production and ion homeostasis, which leads to anoxic depolarisation
(Rossi et al., 2007). These events are followed by a massive release of glutamate in the extracellular space, which is responsible for excitotoxicity on neighbouring cells. The opening of AMPA/KA and NMDA receptors on glial cells causes an increase in intracellular Ca2+ concentrations, which compromises the mitochondrial activity and activates proteolytic enzymes and death pathways. To my knowledge, the results of this section provide the first evidence that mGluR are cytoprotective against hypoxia/ischemia for immature and mature oligodendrocytes in situ. mGluR have been reported to be cytoprotective for
OPCs in kainate- and OGD-induced cell death of OPCs, but not mature oligodendrocytes in vitro (Deng et al., 2004), and in preventing staurosporine- induced apoptosis in OPCs in vitro (Luyt et al., 2006). Similarly, a number of studies have shown that mGluR are cytoprotective for cultured astrocytes against a wide-range of insults, including mGluR5 in OGD-induced apoptosis
(Paquet et al., 2013), mGluR3 in NO-induced cell death (Durand et al., 2010), and group II/III mGluR against LPS-induced death (Zhou et al., 2006), but my results are the first evidence that mGluR are cytoprotective against hypoxia/ischemia for astrocytes in situ. Furthermore, in neurons group I and II mGluR have divergent effects in excitotoxic injury, group II mGluR being neuroprotective (Di
173 Giorgi-Gerevini et al., 2005), whereas group I mGluR can both facilitate and attenuate neuronal death (Bruno et al., 2001). The results of this section show that in glia, activation of both group I and II mGluR is cytoprotective against
OGD-induced cell death. The loss of oligodendrocytes in hypoxia/ischemia is a primary characteristic of PVL or PWMI and is the major cause of death and neurological impairment in cerebral palsy (Banker & Larroche, 1962; Paneth,
1994; Volpe, 1995). Thus, activation of mGluR may be a potential therapeutic target in protecting both astrocytes and oligodendrocytes in PWMI, a possibility supported by a study in which systemic administration of ACPD significantly attenuated the loss of myelin in white matter following hypoxia/ischemia in P6 rats (Jantzie et al., 2010).
5.3.1 Hypoxia/Ischemia effects on astrocytes
My results demonstrate that postnatal optic nerve astrocytes are highly susceptible to ischemia/hypoxia, with a striking halving of their numbers after 60 min OGD, indicative of massive cell death. This is consistent with evidence of ischemia-induced apoptosis in astrocytes (Giffard & Swanson, 2005) and OGD- induced loss of immature astrocytes in the postnatal optic nerve (Fern, 1998;
Thomas et al., 2004). The influx of calcium is one of the most significant events in ischaemia and Ca2+ overload results in mitochondrial dysfunction leading to death (Stys, 2004). In neonatal rat optic nerve astrocytes, the run-down of Na+-K+ pumps and Na+-K+-Cl- cotransport (NKCC1) during hypoxia/ischemia results in a
+ + 2+ rise in [Na ]i and reversal of the Na -Ca exchanger (NCX), resulting in astrocyte
174 cell death (Lenart et al., 2004; Bondarenko et al., 2005). In addition, membrane depolarization and subsequent opening of voltage-operated calcium channels
2+ (VOCC) results in a rise in astrocyte [Ca ]i initially through T-type VOCC within the first 10 minutes of ischaemia, followed by L-type Ca2+ channels (Fern, 1998).
2+ Activation of group I mGluR raises glial [Ca ]i (Section 4), but this clearly does not induce cell death and is cytoprotective for astrocytes in hypoxia/ischaemia. It would be interesting in future studies to examine whether mGluR activation alters Na+ or Ca2+ influx during OGD by interacting with Na+-K+ pumps, NCX, NKCC or VOCC. An interaction between mGluR and VOCC has been described in neurons (Lachica et al., 1995), although in general mGluR activation appears to facilitate Ca2+ influx (Kato et al., 2012).
In addition to these possibilities, mGluR are likely to be protective for astrocytes by regulating multiple downstream elements of cell death pathways
(Vincent & Maiese, 2000; Sugiyama et al., 2007; Paquet et al., 2013). The application of group I and II mGluR agonists had a complete protective effect against the hypoxia/ischaemia induced cell death in optic nerve astrocytes. The group I mGluR prosurvival effect is likely to be mediated via the PI3K/Akt pathway, as there is evidence that mGluR5 mediates the activation of this pathway (Hou & Klann, 2004; Chen et al., 2012), and that oxidative stress in astrocytes is mediated by the regulation of PI3K/Akt, Nrf2 and NF-B pathways
(Shah et al., 2012). A 3h OGD study on cultured astrocytes showed that the activation of mGluR3 also exerts a protective effect by stimulating the PI3K/Atk
175 and ERK1/2 MAPK pathways (Ciccarelli et al., 2007). Another in vitro study investigating the protective effect of mGluR3 in preventing nitric oxide (NO)- induced astrocyte death shows that the activation of this group II mGluR subunit leads to a decrease in cAMP levels, Akt activation and interaction between members of the NF-B family (Durand et al., 2011).
Astrocytes in ischaemia can have a dual role. They can be protective by buffering extracellular potassium and glutamate, scavenging reactive oxygen species and supporting other cells by providing substrates. On the other hand, astrocytes can exacerbate the damage by the propagation of death signals from the infarct core to the penumbra, likely through the generation of aberrant Ca2+ waves, and by the release of toxic substances. Notably, the uptake of glutamate by astrocytes leads to an intracellular accumulation up to very high levels, causing astrocytes to become the main source of glutamate in hypoxic conditions. Hypoxia itself can suppress glutamate transport via deactivation of
NF-B (Dallas et al., 2007). Glutamate-mediated excitotoxicty is a primary cause of neuronal and oligodendrocyte cell death in hypoxia/ischaemia, and impairment of astrocyte glutamate transporters in hypoxia/ischaemia can be prevented by the activation of mGluR1,5 (Liu et al., 2013). Moreover, activated astrocytes upregulate the expression of group I/II mGluR (Aronica et al., 2001) and specifically mGluR5 (Ulas et al., 2000; Ferraguti et al., 2001).
176 5.3.2 Hypoxia/Ischaemia effects on oligodendrocytes
My data on postnatal optic nerves from SOX10-EGFP reporter mice agree with the literature that OPCs and immature oligodendrocytes are highly susceptible to hypoxia/ischaemia, whilst the data from the PLP1-DsRed optic nerves from postnatal and adult mice demonstrates that mature oligodendrocytes are also susceptible to hypoxia/ischaemia, and show for the first time in situ a protective role for group I and group II mGluR. Expression of mGluR3 and 5 has been described in OPCs, immature oligodendrocytes and mature oligodendrocytes in vitro and in vivo, with high levels of mGluR1,5 in
GalC+ oligodenrocytes (Luyt et al., 2006; Jantzie et al., 2010), consistent with my findings in Section 3. In physiology, mGluR are involved in OPC differentiation and myelination (Luyt et al., 2003) and prevention of apoptosis through mGluR5
(Luyt et al., 2006). The function of mGluR in mature oligodendrocytes is unresolved, but in vitro studies suggest a developmental decrease in mGluR5 expression, whereas mGluR3 remain high at all differentiation stages (Luyt et al.,
2006). My results suggest they are protective against hypoxia/ischaemia induced cell death, supporting a study showing a role for group I mGluR activation in the protection of OPCs and immature oligodendrocytes from ischaemic damage, which requires PKC activation (Back et al., 2002; Deng et al., 2004; Luyt et al.,
2006; Jantzie et al., 2010).
Immature oligodendrocytes are more vulnerable than mature oligodendrocytes to oxidative stress (Back et al., 1998), because of their developmental lack of antioxidants (Smith et al., 2000), high levels of expression
177 of AMPA/KA-type iGluR that mediate excitotoxicity (Rosenberg et al., 2003), and because of their increased components of Ca2+ signalling, including VOCC, compared to mature oligodendrocytes (Itoh et al., 2002). Nonetheless, my data demonstrates mature oligodendrocytes are also lost following hypoxia/ischaemia, consistent with a study showing GalC+ oligodendrocytes exposed to 1-2 hours of ischaemia undergo Ca2+-mediated cell death involving
AMPA/KA-type iGluR (McDonald et al., 1998). This mechanism of hypoxia/ischaemia induced cell death has been extensively described in OPCs in vitro (Deng et al., 2003) and immature oligodendrocytes in situ (Fern & Möller,
2000). OPCs and oligodendrocytes express AMPA-, KA- and NMDA-type iGluR and these mediate excitotoxicity in hypoxia/ischemia (Butt et al., 2014a). Hence, the loss of oligodendrocytes in OGD is likely to involve a rise in extracellular glutamate in hypoxia/ischaemia as discussed above, due at least in part to impaired uptake by astrocytes and possibly reversed uptake. The protective effect of mGluR activation on oligodendrocytes may involve an indirect effect via stimulating glutamate uptake in astrocytes (Zhou et al., 2006; Liu et al., 2013).
Furthermore, the stimulation of group I mGluR is known to cause internalization of AMPA- and NMDA-type iGluR in neurons (Snyder et al., 2001; Xiao et al.,
2001). An equivalent mechanism may underlie the protective effect of mGluR on oligodendrocytes, although an in vitro study has shown that the protective effect of group I mGluR in OPCs is not due to iGluR endocytosis (Deng et al., 2004).
Furthermore, there is also evidence that the group II mGluR agonist LY379268 can increase the expression of GluA1 and GluA2 subunits in neurons (Wang et al., 2013), which would counteract the protective effect I observed in
178 oligodendrocytes. Future studies should examine the role of mGluR in internalising iGluR in optic nerve glia and whether this is altered in hypoxia/ischaemia.
Here, I provide evidence supporting a protective role for group I and II mGluR against hypoxia/ischaemia at all stages of oligodendrocyte differentiation.
The protective effect mediated by group I mGluR requires activation of PKC, as demonstrated in vitro in OPCs subjected to kainate exposure and OGD (Deng et al., 2004). PKC is in turn activated by the coupling of Gq protein to PLC. Several G protein coupled receptors (GPCR) are coupled to Gq proteins and my results show that activation of P2Y1 receptors has a protective effect on oligodendrocytes exposed to OGD. Hence, it appears that the stimulation of Gq/11 is prosurvival for oligodendrocytes, irrespective of the GPCR activated. There is evidence supporting that the Gq/11-coupled muscarinic receptors expressed in
CHO cells are able to protect cells from apoptotic cell death mediated by DNA damage (Tobin & Budd, 2003). Further experiments are needed to investigate the mechanisms by which Gq/11 promotes oliogodendrocyte survival, by examining anti-apoptotic factors, such as Akt and Bcl-2, which was not possible in my PhD due to time restraints. Furthermore, depolarization of
+ oligodendrocytes and a consequent rise in [Na ]i results in reversal of the NCX and Ca2+ influx, triggering apoptosis (Stys et al., 1991; Fern et al., 1993). Notably, activation of group I mGluR has been reported to activate NCX in neurons
179 (Hirono et al., 1998; Sekizawa & Bonham, 2006), which requires further investigation.
5.4 Summary and Conclusions
The results of this section provide novel evidence that group I and group
II mGluR protect astrocytes and oligodendrocytes from hypoxia/ischaemia induced cell death in the optic nerve. These studies do not distinguish between direct and indirect effects of mGluR activation in specific cell types, but overall demonstrate that administration of mGluR agonists is glioprotective, consistent with the protective effects of mGluR in various nervous system disorders, including ischaemia-induced brain damage, traumatic brain injury, Huntington's and Parkinson's-like pathology or epilepsy (Flor et al., 2002). Further experiments are required, but the findings of this section indicate that mGluR could be useful therapeutic targets, with the aim of protecting astrocytes, OPCs and oligodendrocytes in case of ischaemic events, such as PVL, predominant in infants and major antecedent of cerebral palsy, and vascular dementia, second most common cause of dementia after Alzheimer’s disease, typically caused by a series of minor strokes.
180
Section 6
Changes in Oligodendroglial Cells in the
Mouse 3xTg Model of Alzheimer’s Disease
181 6.1 Introduction and aims
The results of Section 5 demonstrated that activation of group I/II mGluR agonists protects glia from hypoxia/ischaemia-induced loss, which is implicated in multiple neuropathologies, including stroke, MS, ALS and dementia (Paquet et al., 2013). Specifically, mGluR5 activation is linked with the production of insoluble beta-amyloid (Aβ) and neurodegeneration in Alzheimer’s disease (AD)
(Sokol et al., 2011). mGluR5 acts as a co-receptor for soluble A oligomers, mediating synaptic disruption and dendritic spine loss (Um et al., 2013; Haas et al., 2014). Notably, activated astrocytes are reported to upregulate the expression of mGluR5 in neuropathology (Aronica et al., 2001), and A significantly increased levels of mGluR5 in cultured astrocytes and in the 3xTg-AD mouse model (Ronco et al., 2014). These studies suggest a potential link between mGluR5 and glial changes in ageing that may be altered in AD.
The results of Sections 4 and 5 also demonstrated a functional role for mGluR5 in oligodendrocyte lineage cells, which in relation to AD is of interest, since mGluR5 act as a co-receptor for soluble A oligomers, and there is clear evidence of Aβ toxicity in oligodendrocytes (Xu et al., 2001; Desai et al., 2009).
Indeed, the oligodendrocyte and myelin disruption has been demonstrated as an early feature in AD (Bartzokis, 2002; 2004; Haroutunian et al., 2014), and this is correlated with increased levels of Aβ in human AD (Roher et al., 2004) and animal models of AD (Mastrangelo & Bowers, 2008; Desai et al., 2009).
182 Furthermore, the regeneration of oligodendrocytes throughout life is the function of OPCs (Rivers et al., 2008), and there is evidence that mGluR5 prevent apoptosis of OPC and positively regulate their differentiation into oligodendrocytes (Luyt et al., 2003; Luyt et al., 2006). Hence, mGluR5 expression in oligodendrocyte lineage cells may be related to pathological changes in AD and the capacity of OPCs to regenerate oligodendrocytes throughout life.
However, there is little knowledge on the expression of mGluR5 in AD and very little is known about the changes in OPCs in the ageing brain or in AD.
The aim of this section, therefore, is to examine changes of mGluR5 and
OPCs in AD, using the 3xTg-AD mouse, which harbours PS1 (M146V), APP (Swe) and tau (P301L) transgenes, which has been shown to display astroglial pathology and compromised glutamate homeostasis (Olabarria et al., 2010;
Kulijewicz-Nawrot et al., 2013). To this end, analyses are performed on 6 and 24- months old 3xTg-AD mice and age-matched controls, kindly provided by Prof J.J.
Rodriguez (Bilbao, Spain).
183 6.2 Results
6.2.1 mGluR5 expression in the ageing and 3xTg-AD hippocampus
mGluR5 is widely expressed throughout the brain by neurones and glia, and may be deregulated in AD (Um et al., 2013). Therefore, immunohistochemistry was performed to examine whether mGluR5 expression is changed in the ageing mouse brain and whether this is altered in the 3xTg-AD mouse (Figure 6.1). Immunostaining was characteristically widely distributed and punctate, and it was not possible to resolve specific expression of mGluR5 in the different glial cell types, because of the intense neuronal expression of mGluR5 and close apposition of glia with neurones. However, quantitative analysis of the overall level of immunofluorescence was performed on confocal z-sections, maintaining all parameters the same between experimental groups and subtracting background in negative controls; statistical significance was tested using ANOVA with Newman–Keuls multiple comparison post-hoc analysis. It is important to note that due to a lack of material and time constraints the data for the 24 months 3xTg-AD mouse is based on 3-4 sections from ‘n’=1 mouse and the results for this group are only preliminary, but in all other cases the data are based on 3 or more sections from ‘n’=3 or 4 mice. The results in Figure
6.1 illustrate immunofluorescence results in the stratum oriens, pyramidal layer and stratum radiatum of the CA1 layer of the hippocampus of 6-months and 24- months 3xTg-AD and age-matched controls (Figure 6.1). At the macroscopic level, there was no obvious difference between the experimental groups (Figure
6.1A-D), but higher magnification (Figure 6.1, insets) and immunofluorescence
184 analysis (Figure 6.1E) indicated an age-related decrease in mGluR5 levels between 6- and 24-months in control mice (Figure 6.1A, C, E; p<0.001, ANOVA with Newman–Keuls multiple comparison post-hoc analysis). There was no difference in mGluR5 immunofluorescence in control and 3xTg-AD at 6-months
(Figure 6A, B, E). Interestingly, the preliminary data for the 24-month 3xTg-AD hippocampus indicated that mGluR5 immunofluorescence may be greater compared to age-matched controls (Figure 6.1C, D, E), which requires further analysis for corroboration.
185 Figure 6.1 mGluR5 in the 3xTg-AD mouse hippocampus and age-matched controls. (A- D) Representative confocal micrographs of mGluR5 immunostaining in the CA1 layer of the hippocampus from 6-month and 24-month 3xTg-AD mice and age-matched non-Tg controls (NTG); image acquisition parameters were maintained constant throughout and insets show higher magnification of the stratum radiatum; scale bars = 50 m. (E) Quantification of mGluR5 immunofluorescence intensity in the CA1 measured in constant field of view (FOV, data are mean ± SEM from 3 areas per section, from n=3 or 4 sections per animal, n=3 or 4 animals in all cases except for 24-month 3xTg-AD, where n=1 due to lack of material). ***p<0.001, ANOVA with Newman–Keuls multiple comparison post-hoc analysis. 186 6.2.2 Age-related changes in OPCs in the hippocampus in 3xTg-AD and age-
matched controls
New oligodendrocytes are generated in the adult by OPCs until middle age in humans and this is postulated to be a mechanism by which myelination sustains and enhances cognitive function (Bartzokis, 2011b). However, myelination appears to decline in the ageing brain and studies in the mouse have suggested this is related to a decline in OPCs and/or their regenerative capacity
(Sim et al., 2002). To examine this possibility, immunohistochemical analysis for the NG2 CSPG was used to examine OPCs in the hippocampus of 6- and 24- month old non-Tg (NTG) mice (Figures 6.2-6.5). It is important to note that due to a lack of material and time constraints the data for the 24-month 3xTg-AD mouse is based on 3-4 sections from ‘n’=1 mouse and the results for this group are only preliminary, but in all other cases the data are statistically rigorous and based on analysis of 3 or more sections from ‘n’=3 or 4 mice.
Figure 6.2 illustrates the normal distribution of OPCs in the adult hippocampus from 6-month mice (Figure 6.2A), and high magnification illustrates the characteristic multi-processed morphology of OPCs in the dentate gyrus (DG,
Figure 6.2B), CA1 (Figure 6.2C) and CA3 (Figure 6.2D). In the DG, individual OPC process domains are difficult to distinguish (Figure 6.1B), whilst in the CA1 layer,
OPC process domains overlap only at the edges (Figure 6.1C), and OPCs in the
CA3 display a varying degree of overlap (Figure 6.1D).
187 Similar to stem cells, OPCs self-maintain by undergoing asymmetric divisions generating a mixed progeny of ‘sister cells’ that appear as duplets or triplets when recently divided (Sim et al., 2002). Sister cells either self-sustain
OPCs or proceed to differentiate into oligodendrocytes, hence the frequency of duplets/triplets is an indication of the regenerative potential of OPCs (Boda et al., 2014). To examine this in the 3xTg-AD and ageing mouse, NG2 immunostaining was performed together with nuclear labelling with Hoechst blue to highlight groups of two or more juxtaposed NG2+ sister cells (Figures 6.3 and 6.4). As illustrated for the 6-month non-Tg mouse, sister cells were prominent in all three areas of the hippocampus in the DG (Figure 6.3A), CA1 layer (Figure 6.3B), and CA3 layer (Figure 6.3C). Analysis of age-related changes in NG2+ sister cells shows a significant decrease between 6-month and 24-month in non-Tg controls in the hippocampus overall (Figure 6.4A, C, E; p<0.001, ANOVA with Newman–Keuls multiple comparison post-hoc analysis), and in the DG
(p<0.01) and CA3 (p<0.01), but the decline in OPC sister cells was not statistically significant in the CA1 layer (Figure 6.4E). Compared to age-matched non-Tg controls, the number of NG2+ sister cells was significantly less in 6-month 3xTg-
AD hippocampi overall (Figure 6.4B, E; p<0.001), and in the DG and CA3 (p<0.01), but the decrease was not statistically significant in the CA1 layer (Figure 6.4E).
The number of NG2+ sister cells in the 3xTg-AD hippocampus at 6-months had declined to the level normally seen at 24-months in the non-Tg controls (Figure
6.4B, C, E). At 24-months in the 3xTg-AD hippocampus, the preliminary evidence indicated that the number of NG2+ sister cells is equivalent to age-matched non-
Tg at 24-months (Figure 6.4B, D, E), and to 6-months 3xTg-AD (Figure 6.4C, D, E).
188 The results demonstrated an age-dependent decline in OPC sister cells in the normal ageing hippocampus and that this is accelerated at 6-months in the 3-Tg-
AD mouse.
Astrocytes have previously been shown to undergo cellular changes in the 3xTg-AD model (Rodríguez et al., 2013), hence this was examined in OPCs in the CA1 area, using the Cell Analyst programme (Chvátal et al., 2007) to measure cell surface area and volume in 3xTg-AD and age-matched non-Tg controls
(Figure 6.5). The results showed that there is no difference in the morphological shape and size of OPCs at 6- and 24-months in non-Tg controls (Figure 6.5A, C, E-
H). In contrast, at 6-months OPCs in the 3xTg-AD hippocampi were clearly atrophic compared to age-matched non-Tg controls (Figure 6.5A, B), and showed a significant decrease in cell volume and surface area (see Figure 6E-H for ‘p’ values). Notably, the preliminary evidence from 24-month 3xTg-AD hippocampus indicated OPCs may be hypertrophic compared to age-matched non-Tg controls
(Figure 6.5C, D), and quantification indicated a marked increase in cell surface area and volume compared to 6-month 3xTg-AD hippocampus (Figure 6.5E-H), which requires corroboration.
189 Figure 6.2 Distribution and morphology of OPCs in the hippocampus of 6-month non- Tg mouse. Immunolabelling for the NG2 CSPG was used to identify OPCs. (A) Collage of confocal micrographs illustrating the overall distribution of OPCs in the hippocampus. (B-D) Maximum intensity projections of z-stacks of representative OPCs in the dentate gyrus (B), CA1 layer (C) and CA3 layer (D). Scale bars = 20 m.
190
Figure 6.3 OPC ‘sister cells’ in the hippocampus of 6-month non-Tg mouse. Immunolabeling for NG2 (green) and the nuclear marker Hoechst blue (blue) illustrates the presence of OPC duplets (highlighted by arrows), an indication of recently divided ‘sister cells’. Maximum intensity projections of z-stacks are illustrated in the dentate gyrus (A), CA1 layer (B) and CA3 layer (C), together with single z-sections showing closely juxtaposed duplets (A’, B’, C’) and orthogonal sections through the x-x and y-y planes. Scale bars = 20 m in A, B, C and 10 m in A’, B’, C’.
191 Figure 6.4 Age-dependent changes in OPC sister cells in 3xTg-AD hippocampus. (A-D) Representative images of immunostaining for NG2 (green) and the nuclear stain Hoechst blue (blue) in the DG of 6-month and 24-month 3xTg-AD and age-matched non-Tg (NTG) mice; asterisks indicate sister cells; scale bars=50 m. (E-H) Number of sister cells per constant field of view (FOV) in the hippocampus overall (E), and the DG (F), CA1 (G) and CA3 (H) (data are mean ± SEM from 3 sections per animal, n=3 or 4 animals in all cases except for 24-month 3xTg-AD, where n=1). *p<0.05, **p<0.01, ***p<0.001, ANOVA with Newman–Keuls multiple comparison post-hoc analysis. 192 Figure 6.5 Morphological analysis of OPCs in the 3xTg-AD hippocampus and age- matched non-Tg controls. (A-D) Representative images of OPCs in CA1 of 6- and 24- month 3xTg-AD and age-matched non-Tg (NTG) controls, illustrating in magenta the outline used for analysis of individual cells using the Cell Analyst programme (Chvátal et al., 2007); scale bars=10 m. (E-H) Quantification of OPC total cell volume (E), total cell surface area (F), cell body volume (G) and cell body surface area (H); data are mean ± SEM from 25 cells from 3 sections per mouse, where n=3 or 4 animals in all cases except for 24-month 3xTg-AD, where n=1). *p<0.05, **p<0.01, unpaired t-test analysis.
193 6.2.3 Age-related changes in myelination in 3xTg-AD hippocampus and age-
matched controls
OPCs are believed to generate oligodendrocytes throughout life to replace myelin lost through natural ‘wear and tear’ and to myelinate new connections that develop in response to new life experiences (van Wijngaarden
& Franklin, 2013). The results above indicate that at 6-months in the 3xTg-AD,
OPCs are hypotrophic and there is an apparent acceleration of the age- dependent decrease in sister OPCs, which is implicated in a decrease in their capacity for regenerating oligodendrocytes (Sim et al., 2002; Boda et al., 2014).
In addition, there is evidence of oligodendrocyte/myelin disruption in the 3xTg-
AD mouse brain (Desai et al., 2009; Desai et al., 2010). To examine this, MBP immunostaining was performed to examine age-related changes in myelination in the hippocampus of the 3xTg-AD compared to age-matched non-Tg controls
(Figure 6.6). Analysis of the CA3 layer illustrates a clear age-dependent decrease in the intensity and pattern of MBP immunostaining in non-Tg controls between
6-month (Figure 6.6A) and 24-months (Figure 6.6C), and that this is accelerated in the 6-month 3xTg-AD hippocampus (Figure 6.6B). Quantitative analysis confirmed that MBP immunofluorescence intensity significantly decreased between 6-month and 24-month non-Tg controls (Figure 6.6E; p<0.001, ANOVA with Newman–Keuls multiple comparison post-hoc analysis), and was significantly less in 6-month 3xTg-AD hippocampus compared to age-matched non-Tg controls (Figure 6.6E; p<0.001), equivalent to the level seen in 24-month
194 non-Tg controls. The preliminary data from 24-month 3xTg-AD suggests myelination may decline even further.
195
Figure 6.6 Decrease of MBP immunostaining in the 3xTg-AD hippocampus. (A-D) Representative images of MBP immunostaining in the CA3 layer of the hippocampus of 6- and 24-month 3xTg-AD and non-Tg (NTG) controls mice; scale bars=50 m. (E) Quantification of MBP immunofluorescence intensity per constant FOV (mean ± SEM data from 4 sections per mouse, where n=3 or 4 mice in all cases except for 24-month 3xTg-AD, where n=1). ***p<0.001, ANOVA with Newman–Keuls multiple comparison post-hoc analysis.
196 6.3 Discussion
Previous sections have demonstrated a role for group I mGluR in oligodendrocyte lineage cells, which may be linked to evidence of early changes in myelin in AD (Bartzokis, 2011a) and to studies implicating mGluR5 in AD (Um et al., 2013). The results of this Section do not support early changes in mGluR5 expression in the 3xTg-AD model, but did demonstrate significant age-dependent changes in OPCs that were accelerated in 3xTg-AD and correlated with early disruption of myelin and preceded overt A pathology. Interestingly, preliminary analysis of 24-month 3xTg-AD hippocampus was suggestive of an increase in mGluR5, consistent with evidence that activated glia may upregulate mGluR5 in the 3xTg-AD mouse model (Ronco et al., 2014). However, the key finding of this
Section is of early atrophy of OPCs and a marked decrease in the number of OPC sister cells at 6-months in the 3xTg-AD model, which supports the possibility that a decrease in OPC regenerative capacity may be a key component of oligodendrocyte and myelin disruption that has been demonstrated as an early feature in AD (Bartzokis, 2002; 2004; Haroutunian et al., 2014), and animal models of AD (Mastrangelo & Bowers, 2008; Desai et al., 2009).
6.3.1 The 3xTg-AD model
The results of this section provide evidence of marked changes in OPCs in
3xTg-AD mice. These mice carry the APP Swedish mutations K670N/M671L, the presenilin-1 M146V mutation and the tau P301L mutation in the C57BL6 mice
197 background, and were developed as an AD model by Oddo et al in 2003 (Oddo et al., 2003). This model progressively develops A intracellular deposits and plaques and tau pathology as observed in AD. A deposition starts between 3 and 4 months of age in the neocortex, and after 6 months of age in the CA1 subfield of the hippocampus, in both male and female mice (Rodríguez et al.,
2008). At the same age, 3xTg-AD show altered long term potentiation (LTP) and synaptic dysfunction. My study has been conducted in collaboration with the
Functional Neuroanatomy Laboratory, University of the Basque Country, in
Bilbao, Spain, directed by Prof. JJ Rodriguez, who kindly provided the 3xTg-AD and age-matched control tissue. For my analysis, male 3xTg-AD mice of 6- and
24-months were selected to represent early- and late-stage of the AD pathology.
However, for 24-month 3xTg-AD only one brain was available for analysis within the time available. Nonetheless, the data for the 6-month 3xTg-AD and 6- and
24-month non-Tg controls are from n=3 or 4 mice and are based on a minimum of 3 sections per mouse. Hence, the clearly defined age-dependent changes in
OPCs and myelin and the evidence these are accelerated in 3xTg-AD are statistically reliable.
6.3.2 Changes in OPCs and impact on myelination in 3xTg-AD
OPCs are uniformly distributed in both white and grey matter and are characterised by a complex stellate process arborization, which has been shown to form intimate and multiple contacts with neurones, astrocytes, oligodendrocytes and microglia (Butt et al., 2005). OPCs co-express NG2,
198 PDGFR, and O4, but not mature oligodendrocyte markers (Nishiyama et al.,
1996a; Reynolds & Hardy, 1997), and are the source of oligodendrocytes in the developing and adult brain (Dimou et al., 2008; Rivers et al., 2008; Zhu et al.,
2008b; Kang et al., 2010). Like stem cells, OPCs in the adult brain undergo asymmetric division to generate sister cells that self-sustain OPCs and differentiate into new oligodendrocytes (Dawson et al., 2003; Rivers et al., 2008).
Hence, the frequency of duplets/triplets is an indication of the regenerative potential of OPCs (Boda et al., 2014), which the results of this section show is reduced between 6- and 24-months in the normal mouse hippocampus, consistent with evidence that OPC proliferation rate is significantly reduced with age (Psachoulia et al., 2009). The observation that the overall number of OPCs does not change significantly with age (Rivers et al., 2008) implies that half of the sister OPCs differentiate or die. The numerous NG2+ sister cells observed in the hippocampus is consistent with this being an area of rapid turnover of OPCs, but further studies using proliferative markers and pulse-chase techniques, e.g. with
BrdU and EdU, are required to prove this unequivocally. Nonetheless, there was a significant decrease of number of sister OPCs between 6-months and 24- months in normal non-Tg mice, and the number of sister OPCs at 6-months in
3xTg-AD was significantly less than in age-matched non-Tg mice and equivalent to that observed at 24-months. These results imply the age-dependent decrease in OPC proliferation and regenerative capacity is accelerated in 3xTg-AD, and precedes overt A pathology.
199 In addition, morphological analysis of OPCs highlighted alterations at the early stages of the disease. At 6-months, OPCs in 3xTg-AD displayed a marked atrophy, manifested by a decreased cell volume and surface area compared to
OPCs in non-Tg mice. Furthermore, this was not an accelerated ageing, since the cellular morphology was not altered between 6- and 24-months in non-Tg hippocampus. Although data in the 24-month 3xTg-AD is not statistically reliable, the preliminary evidence indicates that OPC are hypertrophic at later ages of the disease, with increased cell volume and surface area, with thicker processes and enlarged cell body, characteristic of the injury response in OPCs (Ong & Levine,
1999). These results reflect similar changes described in astrocytes by Olabarria et al (Olabarria et al., 2010). The early atrophy of OPCs may be pathologically relevant, considering their role in regenerating oligodendrocytes. Indeed, quantification of myelination using MBP immunostaining indicates myelination is disrupted at 6-months in the hippocampus of 3xTg-AD, comparable to normal
24-month old mice. This supports evidence of myelin disruption previously described in the cortical regions of the 3xTgAD model preceding the appearance of amyloid and tau pathology (Desai et al., 2009). The same group showed that
A is toxic for oligodendrocytes and may be related to AD-related myelin injury in vivo (Desai et al., 2010). Loss of myelin structure and integrity would expose axons to damage, resulting in the decline of cognitive function in the ageing brain. The results of this section support a hypothesis in which accelerated deregulation of OPCs is a major element in the loss of myelin in AD, which is worthy of further study.
200 6.4 Summary and conclusions
The results of this section demonstrate marked age-dependent changes in OPCs that are accelerated in 3xTg-AD and support a hypothesis in which disruption of OPCs and a decline in their regenerative capacity may be a contributor to the loss of myelin in AD. In addition, the results supported the possibility of an age-dependent decrease in mGluR5 in the hippocampus and suggested this may be reversed at 24-months in 3xTg-AD, correlating with A deposition and neuropathological changes in neurons, astrocytes, myelin and
OPCs. Neurotoxic soluble A oligomers lead to neuronal death and memory loss, pathological features of AD. A negatively affects long term potentiation (LTP), the cellular mechanism that underlies learning and memory, and these effects of
A can be reversed with memantine, a non-competitive NMDA-receptor antagonist. mGluR5 is coupled to post-synaptic NMDA receptors, and the application of both NMDA and allosteric mGluR5 antagonists restored LTP in the presence of soluble A in an acute murine hippocampal slice model (Rammes et al., 2011). Moreover, A42, the fragment with the most neurotoxic potential, binds to mGluR5, activating Ca2+ release from intracellular stores (Renner et al.,
2010; Sokol et al., 2011; Um et al., 2013). In turn, mGluR5 activation results in
APP and fragile X mental retardation protein (FMRP) expression increase, mediated by FMRP itself, a RNA binding protein that functions as protein synthesis repressor at synapses (Westmark & Malter, 2007; Malter et al., 2010;
Westmark & Malter, 2012). Another mechanism for A and mGluR5 interaction involves mGluR5 co-expression with A-bound cellular prion protein (PrP(C)),
201 which allows the coupling with Fyn kinases, leading to synaptic disruption (Um et al., 2013). Hippocampal astrocytes of AD patients show higher immunoreactivity for mGluR5 (Lim et al., 2013), which is supported by a study in murine cultured hippocampal astrocytes showing that exposure to A evoked an increase in mGluR5 expression (Grolla et al., 2013). Moreover, reactive astrocytes surrounding A plaques over-express mGluR5, and there is evidence that A binds to astrocytic membrane causing mGluR5 clustering and release of ATP, which slowed mGluR5 diffusion in astrocytes and neurons co-cultured with astrocytes (Shrivastava et al., 2013). My analysis of mGluR5 expression is consistent with these previous findings suggesting an increase in receptor expression in the CA1 layer of 24-month 3xTg-AD mice, but not at earlier stages of the disease. Furthermore, mGluR5 may be involved in OPC differentiation
(Luyt et al., 2003), and in light of the age-related changes in OPCs and myelin in the non-Tg mice and their acceleration in 3xTg-Ad it will be considerable interesting to pursue these studies and investigate changes in the expression and function of mGluR5 in OPCs and their impact on disease progression and myelination.
202
Section 7
General discussion
203 7.1 Summary of key findings
The overall aim of this thesis was to provide insight into the expression and function of mGluR in optic nerve glia, with a focus on its potential glioprotective role in hypoxia/ischaemia. The optic nerve is widely used as a model tissue for studying white matter glia, because it comprises mainly astrocytes and oligodendrocytes, which myelinate and provide support for the axons of retinal ganglion cells, together with small populations of OPCs and microglia (Butt et al., 2004). There is abundant evidence of glutamate-mediated signaling in white matter, but it was unclear whether neurotransmitter signaling mechanisms persist after maturation, and mGluR had not been studied in this context. The key findings of the neurotransmitter profile of optic nerve glia are illustrated in Figure 7.1. It is evident from the results in Section 3 that glutamate receptors are most abundantly expressed in optic nerve glia and the data in
Section 4 confirmed that mGluR in astrocytes and oligodendrocytes are dominated by group I mGluR5, group II mGluR3 and, unexpectedly, group III mGluR4. An important finding is that mGluR were expressed at all ages and were not developmentally downregulated, as had been suggested from previous in vitro studies. A key novel result was that activation of group I and group II mGluR protects both astrocytes and oligodendrocytes from hypoxia/ischaemia -induced cell death. The results of Section 6 supported the possibility of an age-dependent decrease in mGluR5 in the hippocampus, which correlated with a marked age- dependent decrease in OPCs regenerative capacity (Section 6) and of key OPC genes and transcription factors that regulate their differentiation (Section 3).
204 Notably, however, the changes in OPCs and myelin loss are accelerated in 3xTg-
AD, whereas overall mGluR5 expressed did not appear to be decreased. This study provides new insight into mGluR expression and function in white matter glia, and supports a hypothesis in which mGluR are glioprotective and their disruption may be related to the decline in OPCs and their regenerative capacity in the ageing brain. These results are relevant to fundamental glial cell biology and to the pathology of hypoxia/ischaemia, stroke, demyelination and AD.
Figure 7.1 Neurotransmitter profile of the optic nerve.
7.2 Glutamatergic neurotransmission mechanisms in white
matter
The optic nerve does not contain neuronal cell bodies or conventional neuron-to-neuron synapses, and so the prevalence of multiple neurotransmitter receptors may be considered surprising. Notably, adenosine and adrenergic
205 receptors were expressed solely in the postnatal nerve, consistent with a specific role in oligodendrocyte development, but there was a preeminence of glutamatergic, purinergic and GABAergic signaling, in support of previous studies
(Butt et al., 2014a). The physiological importance for glutamatergic signaling in glial cells is well known, ranging from stimulating astroglial homeostatic mechanisms and metabolic support for neurons, to promoting oligodendrocyte development and myelination (see Section 1). Hence, it was not surprising that the genomic profiling of neurotransmitter receptors identified the glutamatergic system as the most highly expressed in the optic nerve (Section 3). Overall, the majority of glutamate receptors were most highly expressed in the postnatal optic nerve, consistent with evidence that glutamate regulates OPC proliferation and differentiation. Notable amongst the developmental changes was the specific expression in postnatal optic nerves of transcripts for the group III mGlur4, which is worthy of further study, since the protein has been detected by immunohistochemistry in retinal ganglion cells axons (Cirone et al., 2002). There have been few studies of mGluR4 in glial cells, but it has been shown to be expressed in reactive astrocytes at edges of MS lesions, but not microglia/macrophages (Geurts et al., 2005), as well as in cultured astrocytes
(Yao et al., 2005), although mGluR4 agonists did not alter cAMP in astrocytes, indicating they were not functional (Prezeau et al., 1994; Wroblewska et al.,
1998). There is also pharmacological evidence for group II mGluR in cultured microglia (Pinteaux-Jones et al., 2008; McMullan et al., 2012).
206 The source of glutamate in the optic nerve is likely to be axonal and to an unknown extent also astroglial. There is evidence of glutamate-loaded vesicles in premyelinated optic nerve axons (Alix et al., 2008), and propagation of action potentials along premyelinated axons in the optic nerve and corpus callosum leads to a rapid vesicular release of glutamate (Kukley et al., 2007; Ziskin et al.,
2007). Astrocytes are also capable of vesicular neurotransmitter release (Kreft et al., 2009; Zorec et al., 2012) and represent a potential source of neurotransmitters. This is supported by findings in Section 3, which highlighted the expression of the axonal transcripts for synaptic vesicles (Sv2b and Syt1), as well as proteins of the SNAP-SNARE complex, which is responsible for vesicular release in both axons and astrocytes (Verkhratskiĭ & Butt, 2013). An interesting finding was that the expression of the transcripts for synaptic proteins switch from SNAP23/Synaptobrevin2/Synaptotagmin4/syntaxin6,7 in the young nerve to SNAP25/Cellubrevin/Synaptotagmin4/syntaxin12 in the ageing optic nerve
(Figure 7.2). The possible functional significance of this switch is unclear and further studies are required to compare mRNA and protein expression, using qRT-PCR and western blot, together with localization of the proteins in axonal and glial compartments using immunohistochemistry. In addition, it is possible that these changes are important in age-related changes in white matter integrity, which are poorly defined in rodents but have been reported in humans
(Bartzokis, 2011).
207
Figure 7.2 Age-dependent changes in vesicular apparatus in the optic nerve.
7.3 Purinergic neurotransmission in white matter
The prominence of purinergic signaling in the optic nerve is well reported and it has been considered to be a ubiquitous ‘gliotransmitter’, since astrocytes both release ATP and respond to ATP via a wide range of P2X and P2Y receptors
(Butt, 2011). The genomic profiling in the optic nerve was most interesting because of the low levels of the P2Y1 receptor, which on the basis of pharmacology has been considered the most important in optic nerve signaling
(Hamilton et al., 2008). In addition, my results in Section 5 indicate P2Y1 receptors are protective for oligodendrocytes in hypoxia/ischaemia. Although microarray analysis of mRNA is not a measure of functional protein, previous pharmacological studies used P2Y1 agonists that also act on P2Y12, which my microarray analysis shows to be highly expressed in the postnatal optic nerve and developmentally downregulated. This is of interest, since a decrease in P2Y12 208 is implicated in demyelination (Amadio et al., 2010), which may be relevant to myelin loss or to the failure of remyelination in the ageing brain. In contrast,
P2X7 receptors, which are also implicated in oligodendrocyte/myelin loss in MS and hypoxia/ischaemia (Agresti et al., 2005a; Agresti et al., 2005b; Matute et al.,
2007b; Butt, 2011), were lowest in the aging optic nerve. All cells are a potential source of ATP in injury, but there is evidence that ATP is released under physiological conditions by astrocytes in the optic nerve (Hamilton et al., 2008).
Astrocytes can release neurotransmitters by vesicular and non-vesicular mechanisms, including vesicular release (see above). However, the vesicular nucleotide transporter VNUT was called absent in the optic nerve at all ages, indicating ATP is released by non-vesicular mechanisms, such as via the P2X7 receptor, which was expressed at all ages and has been implicated in ATP release in the optic nerve (Hamilton et al., 2008). The overall decline in P2X7 and other
P2 receptors in the ageing nerve, including the most prominent P2Y12, raises the possibility that deregulation of purinergic signaling may be related to white matter dysfunction in the ageing brain. In this respect, the P2Y13 receptors are also of interest because they are the only P2 receptors increased in the ageing optic nerve and specifically expressed by microglia (McIlvain et al., 2010). These changes in P2 receptors certainly warrant further investigation.
209 7.4 Altered GABAergic and Glutamatergic neurotransmission in
ageing white matter
It is notable, therefore, that I observed a marked increase in the vesicular glutamate transporter VGLUT1 (ALC17A7) in the ageing optic nerve, and to a lesser extent VGLUT2 (ALC17A6), whereas the glial glutamate uptake transporter
GLAST (EAAT1, SLC1A3) was developmentally decreased, raising the possibility that extracellular glutamate levels may be raised in the ageing nerve. In terms of glutamate receptors, mGluR5, mGluR7, and the NMDA receptor subunit GluN2B, were highest in the aging nerve, and the AMPA receptor subunit GluA2 and
NMDA receptor subunits GluN1 and GluN2D were increased in the ageing nerve to levels observed postnatally. These results imply a shift in glutamate signaling in ageing white matter, with an increase in mGluR5, which I show is protective for oligodendrocytes, together with increases in AMPA/NMDA receptors that are implicated in oligodendrocyte cytotoxicity (Matute et al., 2007a). These changes are consistent with white matter disruption in the ageing brain and suggest this may be related to increased susceptibility of oligodendrocytes/myelin to glutamate-mediated cytotoxicity.
Perhaps the most striking change in the ageing optic nerve was the upregulation of a number of GABAAR receptor subunits, which was coupled to an increase in GAD1 and to a lesser extent GAD2, which are the enzymes responsible for catalyzing the production of GABA from glutamate, and were barely expressed at other ages, together with a decrease in the GABA uptake 210 transporters GAT-1 (SLC6A1) and GAT-2 (SLC6A13). Together, these provide evidence of a marked increase in GABAergic neurotransmission in the ageing optic nerve. This is potentially very important in the observed decrease in OPCs and their regenerative capacity in the ageing brain, since OPCs express GABAAR and their function is to inhibit their proliferation (Yuan et al., 1998). It seems reasonable to suggest that the marked changes in OPCs and myelin observed in ageing white matter is related to changes in synaptic signaling and an imbalance between GABAergic and glutamatergic neurotransmission. Oligodendrocytes and
OPCs express NMDA receptors lacking the glutamate binding subunit GluN2
(Pina-Crespo et al., 2010). My microarray analysis shows instead a marked upregulation of GluN2B in the ageing optic nerve, suggesting a functional and age-related change in NMDA-R composition.
7.5 mGluRs are protective for white matter
In section 5, I provided novel evidence that group I and II mGluR protect astrocytes and oligodendrocytes from hypoxia/ischaemia -induced cell death in situ. This function had already been shown in both cell types, mainly for group I mGluR, in several studies in vitro (Back et al., 1998; Fern, 1998; Deng et al., 2004;
Giffard & Swanson, 2005; Luyt et al., 2006; Jantzie et al., 2010). The cytoprotective mechanisms triggered by group I and II mGluR activation in astrocytes and oligodendrocytes are partially known and involve the recruitment of Gq/11 (See section 5), an increase in PKC activity (Deng et al., 2004), and the stimulation of the PI3/Akt pro-survival pathway (Ciccarelli et al., 2007). The
211 cytoprotective effects of mGluR agonists in astrocytes and oligodendrocytes are likely to involve both direct activation of cell survival pathways following activation of mGluR5 and mGlur3 in these cells, and also indirect effects involving other mGluR-mediated mechanisms. For example, hypoxia/ischaemia- mediated loss of astrocytes results in disruption of ion homeostasis, which causes anoxic depolarization (Rossi et al., 2007), leading to massive release of glutamate, mainly from astrocytes, which in turn triggers excitotoxicity in oligodendrocytes by over-activation of AMPA/KA and NMDA receptors (Matute et al., 2007a). Hence, the cytoprotective effect of mGluR5 on astrocytes would alleviate these destructive changes and attenuate oligodendrocyte/myelin loss.
In addition, mGluR5, mGluR2 and mGluR3 mRNA and protein have been detected in cultured microglia (Biber et al., 1999; Taylor et al., 2002; Byrnes et al., 2009). Application of group I mGluR agonists to microglia exposed to lipopolysaccharide (LPS, inflammatory stimulus) inhibits microglial activation, production of NO and reactive oxygen species, and neurotoxicity (Byrnes et al.,
2009). A recent study has shown that activation of mGluR3 was protective in a model of myelin-induced neurotoxicity(Taylor et al., 2002; Pinteaux-Jones et al.,
2008). Thus, the results of this study show that modulation of mGluR represents a multipotential therapeutic strategy to decrease hypoxia/ischaemia-mediated white matter destruction, which is relevant to hypoxia/ischaemia, cerebral palsy, stroke, multiple sclerosis, neurodegenerative diseases and dementia.
212 7.6 Link between ageing and hypoxia
Aging is characterized by decreased oxygen supply to tissue, a reduction of tissue PO2 and of the activity of several enzymes and metabolic factors. Nonetheless, a reduction of cells' oxygen supply is concomitant to a parallel decrease in oxygen demand by tissues. Because hypoxia per se modulates mitochondria activity, influencing oxygen consumption, hypoxia and aging could share some link. Ageing can be defined as a decline of body functions, due to several factors that affect multiple systems in the brain such as memory and learning processes (Tapia-Arancibia et al., 2008).
Over time, different theories proposed a variety of basic mechanisms that govern ageing. The most popular of these theories was the Mitochondrial Theory of Aging (MTA), first proposed by Harman in 1956 (Harman, 1956). According to this theory, aging results from the accumulation of oxygen free radical [reactive oxygen species (ROS)] damage to cellular components, including nucleic acids.
Over the years, the theory was refined by, first, the identification of mitochondria as both the source and the target of the ROS (Harman, 1972), and, then, the identification of mitochondrial DNA (mtDNA) as most susceptible target of the damage (Fleming et al., 1982; Miquel et al., 1983). Damage to mtDNA can be converted to point mutations and deletions, which can be transmitted to and accumulated in daughter molecules through the process of replication, enabling deterioration of the integrity of hereditary information over time.
Under normal physiological conditions, ROS emission (essentially, production minus scavenging) is considered to account for 0.25-11% of the total 213 oxygen consumed by mitochondria, depending on the animal species and respiration rates (Aon et al., 2012). When ROS production is not compensated for with their scavenging by endogenous antioxidants, ROS will rise beyond the physiological threshold level, causing a process conventionally called “oxidative stress”. The reaction of formation of a primary ROS (superoxide only) generated in the respiratory chain from molecular oxygen is dependent on oxygen concentration. Oxygen supply influences cell metabolism and is a key factor in regulating cell growth, and thus, the oxygen-gradient diffusion at capillary tissue level is essential for the cellular survival. In hypoxic conditions, ROS are generated. Paradoxically, the generating ROS in mitochondria in the cell remains constant or even increases when PO2 drops dramatically. Elevated mitochondrial
ROS generation in the cell in response to hypoxia is not intrinsic to the mitochondrial respiratory chain alone but can be attributed to some involvement of extramitochondrial factors (Hoffman et al., 2007). For example, hypoxia- induced superoxide production can occur through activation of NADPH oxidase located in the cell membrane (Marshall et al., 1996). In addition, NO synthesis in mitochondria can partially block cytochrome oxidase (Brown & Cooper, 1994;
Schweizer & Richter, 1994), thus reducing mitochondrial electron carriers and favoring generation of superoxide at hypoxic conditions (Turrens, 2003).
In my study, Cox7a2 was one of the genes most expressed in the 18 months old optic nerve (Section 3, Table 3.7). Cox7a2 encodes for a subunit of
Complex IV, cytochrome oxidase and its expression is upregulated in cardiac tissue after hypoxic exposure (Everett et al., 2012). Gene expression during hypoxia is regulated by the HIF transcription factor family (Kenneth & Rocha,
214 2008). Under normal oxygen conditions, a subunit of the HIF transcription factor is hydroxylated by prolyl hydrolases, and subsequently rapidly degraded.
However, during hypoxia the protein stabilizes and associates with its other subunits and enters the nucleus where it interacts with cofactors and can affect transcription (Kenneth & Rocha, 2008). Although the HIF gene didn’t stand out in the microarray study presented in Section 3, it would be interesting to examine its expression in the old optic nerve tissue and, in light of the glio protective properties of mGluRs activation shown in Section 5, investigate the correlation between the metabotropic glutamate receptors and HIF in astrocytes and oligodendrocytes.
7.7 Myelination and oligodendrocytes in ageing and pathology
The optic nerve microarray analysis showed a decrease in oligodendrocytes and OPC markers in ageing tissue compared to postnatal and adult optic nerve. The difference does not appear to be due solely to the fact that the postnatal tissue is actively myelinating, because OPC differentiation factors such as Pdgfra, Sox10, Olig2 and Nkx2.2 were also downregulated.
Moreover, genes associated with transcription, translation and protein folding were increased, suggesting an alteration in the maintenance of the homeostasis of the proteasome that is associated with ageing and protein aggregation- diseases (Hipp et al., 2014). These results suggest there might be a link between ageing, impairment of OPC proliferation and loss of myelin, which is considered a
215 hallmark of cognitive impairment associated with the major neurodegenerative diseases such as AD (Sim et al., 2002; Bartzokis et al., 2003; Bartzokis, 2004).
Thanks to a collaboration with Prof Rodriguez (Bilbao, Spain), I had the opportunity to test this hypothesis on brain tissue of 3xTg-AD mice.
Unfortunately, due to lack of material and time, the data on the 24-month old group are only preliminary and have no statistical relevance. Nonetheless, I found significant age-dependent differences in adult OPCs in the other age- groups, which were statistically valid. Adult OPCs undergo asymmetric division, giving rise to sister cells (Dawson et al., 2003; Rivers et al., 2008), and my results demonstrated a significant decrease in the number of sister cells at 6-month in
3xTg-AD, comparable to that observed in 24-month non-Tg age-matched controls. The results support evidence that OPC cell division slows with age
(Psachoulia et al., 2009), and this is accelerated in 3xTg-AD, prior to the onset of overt A pathology. A key feature of OPCs is that they form synapses with neurons, both glutamatergic and GABAergic (Bergles et al., 2000; Lin & Bergles,
2004) and this is proposed to regulate their proliferation and hence capacity for regenerating oligodendrocytes (Sim et al., 2002). Deregulation of synaptic signaling precedes overt A neuropathology in 3xTg-AD, reflecting the situation in human AD (Desai et al., 2009; Desai et al., 2010), and my results demonstrated deregulation of glutamatergic and GABAergic neurotransmission in ageing white matter. Taken together, these studies put forward a hypothesis that early deregulation of synaptic signaling results in a decrease in OPC regenerative capacity, contributing to the age-dependent loss of myelin and decline in cognitive function. Future studies could investigate this in mouse models of AD. 216 7.8 Conclusions
In conclusion, this PhD thesis has provided insights into the neurotransmitter profile of the optic nerve and how it changes with age. I have provided evidence supporting the functional expression of mGluRs in glia and determined a cytoprotective role for mGluR5 and mGluR3 in astrocytes and oligodendrocytes subjected to hypoxia/ischaemia in situ. Lastly, I provided evidence of early changes in OPCs in 3xTg-AD. Taken together with the genomic profiling and functional studies on mGluR, this thesis supports a role for glial neurotransmission in regulating developmental oligodendrocyte differentiation and myelination, and suggests deregulation of neurotransmission in the ageing brain may be linked to oligodendrocyte/myelin loss and white matter destruction. Thus, my thesis provides evidence that mGluR are a potential therapeutic target for maintaining white matter integrity in pathology and in ageing.
217
Section 8
Appendix
218
Figure 8.1 Relationship between OPCs and A in 3xTg-AD. Confocal images of coronal brain sections from 24-month 3xTg-AD double immunostained for NG2 (green) and A (red). (A) Overview of the association of OPCs with A in the CA1 and CA3 layers of the hippocampus. (B,C) Deposition of A in the cortex and close association of OPCs with Aβ containing neurones (B, B’) and blood vessels (C, C’). (D, D’) OPCs closely associated with neurones containing Aβ in the basolateral amygdala nucleus. Scale bars = 100 m in A-D, 10 m in B’ and 20 m in C’ and D’.
219
Figure 8.2 Relationships between OPCs and A plaques in the hippocampus of 24- month 3xTg-AD. Confocal images of CA1 hippocampus from 24-month 3xTg-AD mouse double immunostained for NG2 (green) and A (red). (A) Overview of relations between OPCs and Aβ plaques in the CA1 region. (B-D) Intimate association of OPCs with A plaques in maximum intensity z-stack projection (B), single z-section and orthogonal x-x and y-y sections (C), and isoformed 3-D view (D). (E) Maximum intensity z-stack projection of OPC located between four A plaques. (F) OPC embedded within an Aβ plaque in maximum intensity z-stack projections (F, F’’’ and F’’’’) and orthogonal x-x and y-y sections (F’, F’’). Scale bars = 50 m in B and 20 m in A-A’’ and B’-F’’’’ 220
Figure 8.3 Astrocytes distribution and morphology in the hippocampus of 6-month non-Tg control. GFAP immunostaining illustrating the overall distribution of astrocytes in the hippocampus (A) and maximum intensity z-stack projections of representative GFAP+ cells in the dentate gyrus (B), CA1 layer (C) and CA3 layer (D). Scale bars = 100 m in A and 20 m in B-D.
221
Figure 8.4 Interrelations between OPCs and astrocytes with A plaques in the 24- month 3xTg-AD hippocampus. Confocal images of CA1 region triple immunostained for NG2 (green), GFAP (red) and A (blue). (A) OPCs and astrocytes are uniformly distributed in the CA1 layer with overlapping process domains. (B) OPCs and astrocytes are associated with the same Aβ plaques. (C-E) Higher magnification maximum intensity z-stacks showing OPCs and astrocyte processes intertwined within Aβ plaques (C), together with single z-section (D) and orthogonal x-x and y-y sections showing juxtaposition of their processes within the A plaque (D’-D’’). (E) Juxtapostion of OPCs and astrocytes in the absence of Aβ deposits. (F, G) Maximum intensity z-stack projection of a large A plaque containing cells co-expressing NG2 and GFAP (F, colocalization appears yellow) and higher magnification of individual cell (G). Scale bars = 100 m in A, 50 m in B’, 20 m in C, D and F, and 10 m in E and G
222
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