1,721,103 research outputs found
In utero exposure to maternal obesity and hyperglycaemia and the fetal brain epigenome
Get PDFOffspring development is particularly vulnerable during the antenatal period and exposure of the fetus to adverse environments in utero during this critical period may impact on their health trajectories and disease susceptibility in the long term. This phenomenon, known as the “developmental origins of health and disease”, was described by David J Barker approximately 30 years ago. Further human and animal studies on fetal programming continued to support the pivotal role of the intrauterine environment in influencing the offspring phenotype and this transgenerational relationship may be mediated by modifications to the offspring epigenome.
Overnutrition and obesity during pregnancy has become a significant problem, mirroring the global obesity epidemic. Obesity during pregnancy is associated with an increased risk of maternal complications during the perinatal period, including gestational diabetes mellitus (GDM). Offspring born to obese and/or diabetic mothers are susceptible to short- and long-term morbidity and mortality, including an increased risk of obesity and cardiometabolic disorders in later life. Additionally, there is emerging evidence indicating an increased prevalence of offspring neurodevelopmental disorders and impaired cognitive function following exposure to maternal obesity and/or diabetes, although the mechanisms remain unclear.
I hypothesise that exposure of the developing brain to maternal obesity and diabetes during the antenatal and early postnatal period impacts on the epigenetic landscape, specifically DNA methylation, with downstream effects on genes associated with critical neurodevelopmental processes. I utilised a murine high fat diet-induced obesity in vivo model and demonstrated that exposure of dams to high fat high sugar diet (HFHS) recapitulated the phenotype seen in obese human pregnancies. Further, I showed that the HFHS offspring were hypoglycaemic and small-for-gestational age, which are known offspring complications associated with maternal obesity and hyperglycaemia during pregnancy. The expression of key enzymes involved in DNA methylation, specifically the Ten Eleven Translocase (TET) and DNA methyltransferase (DNMT) enzymes, were altered in male, but not female, HFHS offspring cortex and cerebellum, Additionally, I identified a sex-specific difference in cerebellar 5mC and 5hmC patterns, a novel finding that has not been described elsewhere. I have also demonstrated that exposure to maternal obesity and hyperglycaemia in the antenatal period associated with perturbations in metabolic and cell differentiation pathways.
Using an in vitro mixed-species cell culture system, I explored the impact of exposure of altered glucose availability on the transcriptomic and epigenetic landscape, as well as metabolic activity of 2 of the key cell types in the developing brain – astrocytes and neurons. This study confirmed that in silico analysis of mixed species astrocyte-neuron co-culture following MeDIP-sequencing was possible. I demonstrated that while exposure to high glucose concentrations had a modest impact on astrocyte and neuron transcriptome, there was evidence of perturbations in DNA methylation patterns, particularly in neurons. I identified that exposure of neurons to hyperglycaemic conditions impacted on differential methylation of promoters associated with cell signalling and synaptic function in neurons. I have also shown that changes in glucose availability impacts on mitochondrial respiration in both neurons and astrocytes.
Finally, using the in vitro cell culture model, I interrogated the non-cell autonomous regulation of the methylation and transcriptional landscape in astrocytes and neurons. I identified that astrocytes impact on neuronal DNA methylation with downstream effects on the transcription of key genes involved in neuronal synaptic activity and development, including immediate early response genes (Fos, Egr1 and Dusp5), as well as genes involved in key metabolic processes (Sucla1 and Pdk1). Further, the presence of neurons impacted on DNA methylation patterns and gene expression of a key astrocyte glutamate transporter, Slc1a2.
Collectively, the findings from this thesis highlights the role of epigenetic modification – specifically DNA methylation - in influencing the gene expression and pathways associated with key neurodevelopmental processes following the exposure to maternal obesity and diabetes. It has also demonstrated that altered glucose availability associates with changes in astrocyte and neuron transcriptome, methylome and energetics. Additionally, it has also contributed to our current knowledge of the non-cell autonomous relationship between astrocytes and neurons
Control of anti-apoptotic and antioxidant pathways in neural cells
Get PDFOxidative stress is a feature of many chronic neurodegenerative diseases as well as a
contributing factor in acute disorders including stroke. Fork head class of
transcription factors (Foxos) play a key role in promoting oxidative stress-induced
apoptosis in neurons through the upregulation of a number of pro-apoptotic genes.
Here I demonstrate that synaptic NMDA receptor activity not only promotes Foxos
nuclear exclusion but also suppresses the expression of Foxo1 in a PI3K-dependent
fashion. I also found that Foxo1 is in fact, a Foxo target gene and that it is subject to
a feed-forward inhibition by synaptic activity, which is thought to result in longerterm
suppression of Foxo downstream gene expression than previously thought. The
nuclear factor (erythroid 2-related) factor 2 (Nrf2) is another transcription factor
involved in oxidative stress and the key regulator of many genes, whose products
form important intrinsic antioxidant systems. In the CNS, artificial activation of Nrf2
in astrocytes has been shown to protect nearby neurons from oxidative insults.
However, the extent to which Nrf2 in astrocytes could respond to endogenous signals
such as mild oxidative stress is less clear. The data presented herein, demonstrate for
the first time that endogenous Nrf2 could be activated by mild oxidative stress and
that this activation is restricted to astrocytes. Contrary to the established dogma, I
found that mild oxidative stress induces the astrocytic Nrf2 pathway in a manner
distinct from the classical Keap1 antagonism employed by prototypical Nrf2
inducers. The mechanism was found to involve direct regulation of Nrf2's
transactivation properties. Overall these results advance our knowledge of the
molecular mechanism(s) associated with the control of endogenous antioxidant
defences by physiological signals
Molecular mechanisms of neuronal homoeostasis in vivo
Get PDFHomeostatic plasticity is important in neurobiology for stabilising neuronal networks
in the face of Hebbian forms of synaptic plasticity that are thought to mediate memory
storage. Impairment of homeostatic plasticity has also been implicated in neurological
diseases such as Rett syndrome and fragile X syndrome. Homeostatic plasticity
can be achieved through scaling of the strength of synaptic connections between
neurones or by changes in intrinsic excitability. While homeostatic plasticity has been
studied mainly using in vitro preparations, it is for the most part not known whether
changes of neural activity in vivo induce homeostatic changes. The molecular pathway
responsible for homeostatic plasticity still remains unclear.
In this thesis, I have used stereotaxic surgery to over express Kir2.1, an inwardly
rectifying potassium channel, in vivo in the brains of adult mice. I show that the expression
of Kir2.1 through adeno-associated virus (AAV) does not cause any adverse
effects in the dentate gyrus nor the CA1 of the mouse hippocampus. I go on to use
slice patch clamp methods to measure the change in electrical properties of granule
cells in the dentate gyrus and pyramidal cells in CA1 caused by expression of Kir2.1.
I show that the excitability of neurones expressing Kir2.1 was reduced compared to
control neurones. By 2 weeks after virus injection the neurones showed homeostatic
plasticity in response to Kir2.1 over expression. Interestingly, the mechanism of adaptation
was different in different types of cells; dentate gyrus granule cells adapted
through change in their intrinsic excitability, whereas CA1 pyramidal cells adapted
by modifying the strength of their synaptic inputs.
To establish whether induction of homeostatic plasticity is associated with changes
in gene expression I used fluorescent activated cell sorting (FACs) to isolate pure
population of neurones infected with viruses. I then sequenced RNA extracted from
neurones expressing Kir2.1 and control neurones. Analysis of the RNAseq data revealed
molecular candidates involved in homeostatic plasticity.
In summary, I show that Kir2.1 over expression causes change in excitability and
subsequent homeostatic plasticity in vivo. The mechanism of adaptation differs between
cell types. RNAseq results identify novel candidates for future investigation
Cellular and molecular insights into neurodegeneration mediated by the C9orf72 repeat expansion mutation
Get PDFAmyotrophic lateral sclerosis (ALS) is an incurable, rapidly progressive and fatal neurodegenerative disorder, characterised by loss of upper and lower motor neurons (MNs). Approximately 10-20% of ALS cases are familial, of which the C9orf72 (G4C2)n>30 hexanucleotide repeat expansion mutation is the commonest known cause in the western world. The finding that familial ALS is clinically and pathologically indistinguishable from sporadic ALS supports the study of monogenetic causes to better understand common pathogenic mechanisms. Thus, human induced pluripotent stem cell (iPSC) derived MN experimental platforms, combined with paired gene-edited isogenic control lines, provide a powerful approach, both to identify early-stage disease-driving mechanisms, and establish causality between a given mutation and phenotypes. Such in vitro disease modelling is complemented by in vivo approaches, particularly those that harness technologies interrogating molecular neuropathological signatures in human post-mortem tissue.
The primary aim of this PhD was to study the MN cell-autonomous pathogenic mechanisms of the C9orf72 repeat expansion against an isogenic control background using a human model, adopting cellular, molecular and bioinformatics techniques.
I establish that C9orf72 MNs have dysfunctional axonal homeostasis, with aberrations both in their axonal morphology (reduced neurite length) and function (impaired fast axonal transport of mitochondrial cargo). Axonal dysfunction is a common phenotype in neurodegenerative disorders, including in ALS, where MNs have axons extending up to a metre long. The maintenance of axonal function is a highly energy-demanding process, raising the question of whether MN cellular energetics is perturbed in ALS, and whether its recovery promotes axonal rescue. I show that these axonal phenotypes are associated with concomitant metabolic dysfunction, owing to defective mitochondrial respiration. Unbiased RNA-sequencing revealed reduced expression of electron transport chain transcripts, encoded by mitochondrial DNA, the copy number of which was unaltered. Critically, I show, through neuropathological analysis of patient post-mortem tissue, that this transcriptomic dysregulation is selective to anterior horn spinal (motor) neurons, and is absent in dorsal horn spinal (sensory) neurons, with corresponding alterations reflected at the level of protein expression. Through manipulation of this molecular mitochondrial loss-of-function signature, leading to therapeutic rescue of the observed dysfunctional axonal homeostasis, I determine a novel causal relationship between axon dysfunction and contributory metabolic dysfunction in C9orf72-ALS.
Collectively, my experimental data show that loss of mitochondrial function is a key mediator of axonal dysfunction in C9orf72-ALS, and that boosting MN bioenergetics is sufficient to restore axonal homeostasis, opening new potential therapeutic strategies for ALS that target mitochondrial function. These data build on preclinical data from animal models in ALS (albeit largely biased by a preponderance of data from SOD1 models), which I systematically reviewed and meta-analysed, showing that targeting mitochondrial dysfunction may prolong survival, particularly if the intervention is administered early
Cortical development & plasticity in the FMRP KO mouse
Get PDFAutism is one of the leading causes of human intellectual disability (ID).
More than 1% of the human population has autism spectrum disorders (ASDs), and it
has been estimated that over 50% of those with ASDs also have ID. Fragile X
syndrome (FXS) is the most common inherited form of mental retardation and is the
leading known genetic cause of autism, affecting approximately 1 in 4000 males and
1 in 8000 females. Approximately 30% of boys with FXS will be diagnosed with
autism in their later lives.
The cause of FXS is through an over-expansion of the CGG trinucleotide
repeat located at the 5’ untranslated region of the FMR1 gene, leading to
hypermethylation of the surrounding sequence and eventually partially or fully
silencing of the gene. Therefore, the protein product of the gene, fragile X mental
retardation protein (FMRP), is reduced or missing.
As a single-gene disorder, FXS offers a scientifically tractable way to
examine the underlying mechanism of the disease and also shed some light on
understanding ASD and ID. The mouse model of FXS (Fmr1−/y mice) is widely
accepted and used as a good model, offering good structural and face validity. Since
a primary deficit of FXS is believed to be altered neuronal communication, in this
thesis I examined white matter tract and dendritic spine abnormalities in the mouse
model of FXS. Loss of FMRP does not alter the gross morphology of the white
matter. However, recent brain imaging studies indicated that loss of FMRP could
lead to some minute abnormalities in different major white matter tracts in the human
brain. The gross white matter morphology and myelination was unaltered in the
Fmr1−/y mice, however, a small but significant increase of axon diameter in the
corpus callosum (CC) was found compared to wild-type (WT) controls. Our
computation model suggested that the increase of axon diameter in the Fmr1−/y mice
could lead to an increase of conduction velocity in these animals.
One of the key phenotypes reported previously in the loss of FMRP is the
increase of “immature” dendritic spines. The increase of long and thin spines was
reported in several brain regions including the somatosensory cortex and visual
cortex in both FXS patients and the mouse model of FXS. Although recent studies
which employed state-of-the-art microscopy techniques suggested that only minute
differences were noticed between the WT and Fmr1−/y mice. In agreement with
previous findings, I found an increase of dendritic spine density in the visual cortex
in the Fmr1−/y mice, and spine morphology was also different between the two
genotypes. We found that the spine head diameter is significantly increased in the
CA1 area of the apical dendrites of the Fmr1−/y mice compared to WT controls.
Dendritic spine length is also significantly increased in the same region of the
Fmr1−/y mice. However, apical spine head size does not alter between the two
genotypes in the V1 region of the visual cortex, and spine length is significantly
decreased in the Fmr1−/y mice compared to WT animals in this region.
Lovastatin, a drug known as one of the 3-hydroxy-3-methyl-glutaryl-CoA
(HMG-CoA) reductase inhibitors, functions as a modulator of the mitogen-activated
protein kinases (MAPK) pathway through inhibiting Ras farnesylation, was used in
an attempt to rescue the dendritic spine abnormalities in the Fmr1−/y mice. Mice
lacking FMRP are susceptible to audiogenic seizure (AGS). Previous work has
shown that 48 hr of lovastatin treatment reduced the incidence of AGS in the Fmr1−/y
mice. However, chronic lovastatin treatment failed to rescue the spine density and
morphology abnormalities in the Fmr1−/y mice.
Mouse models are invaluable tools for modelling human diseases. However
inter-strain differences have often confounded results between laboratories. In my
final Chapter of this thesis, I compared two commonly used C57BL/6 substrains of
mice by recording their electrophysiological responses to visual stimuli in vivo. I
found a significant increase of high-frequency gamma power in adult C57BL/6JOla
mice, and this phenomenon was reduced during the critical period. My results
suggested that the C57BL/6JOla substrain has a significant stronger overall
inhibitory network activity in the visual cortex than the C57BL/6J substrain. This is
in good agreement with previous findings showing a lack of open-eye potentiation to
monocular deprivation in the C57BL/6JOla substrain, and highlights the need for
appropriate choice of mouse strain when studying neurodevelopmental models.
They also give valuable insights into the genetic mechanisms that permit experience-dependent
developmental plasticity.
In summary, these findings give us a better understanding of the fine structure
abnormalities of the Fmr1−/y mice, which in turn can benefit future discoveries of the
underlying mechanisms of neurodevelopmental disorders such as ID and ASDs
Investigation into the destructive and adaptive responses of neural cells to stress
Get PDFHomeostasis within the neuro-glial unit is essential to the longevity of neurons.
Conversely, loss of homeostasis, particularly of Ca2+ levels, of redox balance and of
ATP, contribute to neuronal loss and dysfunction in many neurodegenerative and
neurological disorders. This thesis is centred on better understanding the
vulnerability of neurons to stress, as well as adaptive responses to these stresses.
Since neurodegenerative conditions associated with Ca2+, redox and bioenergetic
dyshomeostasis are often characterised by early dendritic pathology, I first studied
dendritic vs. somatic responses of primary cortical neurons to these types of
challenges in real-time. Using a wide range of genetically-encoded probes to
measure Ca2+, ATP, NADH, glutathione and glutamate, I show that dendrites are
selectively vulnerable to oxidative stress, excitotoxicity as well as to metabolic
demand induced by action potential (AP) burst activity. However, I provide evidence
that neurons undergoing energetically demanding AP burst activity can adjust their
metabolic output by increasing mitochondrial NADH production in a manner
dependent on the mitochondrial calcium uniporter (MCU), as well as increase their
capacity to buffer their intracellular redox balance. Finally, I have studied
transcriptional programs in astrocytes triggered by neurons and neuronal activity to
better understand adaptive signaling between different cell types in the neuro-glial
unit. I developed a novel system combining neurons and astrocytes from closely-related
species, followed by RNA-seq and in silico read sorting. I uncovered a
program of neuron-induced astrocytic gene expression which drives and maintains
astrocytic maturity and neurotransmitter uptake function. In addition I identified a
novel form of synapse-to-nucleus signaling, mediated by glutamatergic activity and
acutely regulating diverse astrocytic genes involved in astrocyte-neuron metabolic
coupling. Of note, neuronal activity co-ordinately induced astrocytic genes involved
in astrocyte-to-neuron thyroid hormone signaling, extracellular antioxidant defences,
and the astrocyte-neuron lactate shuttle, suggesting that this non cell-autonomous
signaling may form part of the homeostatic machinery within the neuro-glial unit
Contribution of the centriolar protein Trichoplein to endothelial cell function in brain vasculature
Get PDFAge-related cerebrovascular dysfunction plays a critical role in the pathogenesis of cerebrovascular disease, vascular dementia and Alzheimer’s disease, but therapeutic development has been largely unsuccessful until now. Endothelial cells (ECs) are a fundamental component of the neurovascular unit. Their dysfunction has been established as an early event in the pathogenesis of cerebrovascular disease and vascular dementia, leading to dysregulation of cerebral blood flow and blood-brain barrier damage. In this context, identifying novel genes associated with endothelial dysfunction will help understand the role of ECs in blood-brain barrier integrity and address new therapeutic targets.
Trichoplein (TCHP) was initially characterised as a ubiquitously expressed keratin filament-binding protein associated with cell division and cilia formation. Moreover, TCHP has been reported to regulate ER-mitochondria tethering and promote mitophagy, a specialised form of autophagy necessary for the turnover/remodelling of mitochondria. In the lab, we previously demonstrated a pivotal role for the centriolar protein TCHP in linking endothelial cell function with the control of autophagy, showing that the depletion of TCHP in ECs impairs migration and sprouting and triggers cellular inflammation. In line with this, the endothelial-specific deletion of Tchp (TchpEC) in mice decreased the blood flow recovery and vascularisation following hind-limb ischaemia. Protein aggregates were detected in ECs from TchpEC mice and ECs from patients with coronary artery disease.
However, the presented preliminary data regarding the role of TCHP in brain vasculature has not been explored yet.
My PhD project aims to characterise the role of TCHP in brain microvascular ECs in vitro and in vivo and to reveal its role in blood-brain barrier integrity.
For this study, I generated mice with endothelial selective Tchp knock-out (TchpEC ) by breeding the conditional knock-out mice with mice carrying Cre recombinase under the VE-cadherin promoter. RNA sequencing demonstrated the up-regulation of matrix-metalloproteinases and chemokine signalling pathways in the brain ECs isolated from TchpEC mice. The analysis of the in vitro permeability by Electric Cell-substrate Impedance Sensing (ECIS®) revealed an impaired barrier function in ECs lacking TCHP. Furthermore, TchpEC mice administered with the fluorescently labelled tracer dextran presented a higher tracer accumulation in the brain than WT mice, showing a loss of blood-brain barrier integrity. In addition, the presence of protein aggregates was confirmed in the cytoplasm of brain microvascular ECs lacking TCHP. The proteomic characterisation of the insoluble- protein fraction revealed RNA-binding and proteasome-associated proteins, suggesting the toxicity of these aggregates for the cells.
Finally, a pharmacological screening identified an FDA-approved compound activating autophagy and, thus, restoring EC function and reducing expression of inflammatory genes in EC lacking TCHP.
Collectively, this study presents the novel role played by TCHP in the cerebrovascular endothelium and identifies a new mechanism by which the silencing of TCHP could link endothelial dysfunction to impaired blood-brain barrier integrity
There and back again: functional outcomes of reciprocal neuron-astrocyte signalling
Get PDFNeurons do not exist in isolation in the central nervous system, and there is a growing appreciation that the interactions between neuronal and non-neuronal cells are fundamentally important for nervous system function. A major family of non-neuronal cells are the astrocytes, with a surge of recent work suggesting the relationship between neurons and astrocytes is bidirectional and highly complex. In my thesis I seek to further uncover the nature of this intimate relationship between neurons and astrocytes of the cortex. One well-established role of astrocytes is the collection of neuronal glutamate via their high affinity excitatory amino acid transporters, with dysfunctions in this system being linked to numerous neurological diseases. Previous reports suggest that neurons may regulate the expression of these astrocytic glutamate transporters, through an as yet unknown pathway. In my thesis I first investigate the nature of this non-cell-autonomous neuronal control of astrocytes. I begin by using results from the lab’s novel mixed-species RNA-sequencing dataset to explore how neurons regulate astrocytic gene expression, finding that they upregulated the astrocytic glutamate transporters. By electrophysiological recording I show a corresponding functional increase in the astrocytes’ ability to collect glutamate, before demonstrating that neurons upregulate the astrocytic transporters through Notch signalling. I then investigate whether continuous Notch signalling is required to maintain these transporters’ expression and function, finding that removal of Notch signalling after the establishment of transporter expression significantly reduces the transporters’ activity. For the remainder of my thesis I explore how cortical astrocytes may in turn control cortical neuronal function. Using RNA-seq data generated in the lab I discover a host of neuronal genes that are regulated by astrocytes. Amongst these genes were the functionally important K+ inward rectifying channel family, which were strongly downregulated in neurons by astrocytes, an observation hitherto unseen. I hypothesise that this downregulation will result in alterations to neuronal membrane properties which will enhance neuronal excitability, and that this may in turn have down-stream consequences on neuronal activity and synaptogenesis. I find that cortical neurons are rendered more excitable by astrocytes, leading to an enhancement of neuronal activity, driven by the astrocyte-induced decrease in K+ inward rectifiers. Although I do not see an increase in baseline synaptogenesis, I show a range of homeostatic neuronal responses emerge in the presence of astrocytes. This work suggests that astrocytes play a central role in regulating neuronal activity
Investigation of inflammatory and oxidative stress mechanisms in the disruption of white matter structure and function following chronic cerebral hypoperfusion
Get PDFVascular cognitive impairment (VCI) describes a heterogeneous condition caused
by cerebrovascular disease and disturbances in cerebral blood flow delivery. It is the
second leading form of dementia and vascular factors such as hypertension, diabetes
and obesity are associated with an increased risk of developing VCI. White matter
alterations are a prominent pathological feature observed in patients with VCI thought
to underlie cognitive impairment. Neuroimaging studies show a positive correlation
between the burden of white matter alterations and progressive cognitive impairment.
Similarly associated both with white matter alterations and cognitive impairment is
chronic cerebral hypoperfusion, sustained subtle reductions in cerebral blood flow.
Cerebral hypoperfusion is observed before the onset of cognitive decline in humans
and reducing cerebral blood flow in animal models replicates important aspects of
VCI, suggesting hypoperfusion is an early driver of white matter disruption and VCI.
Human neuropathology and preclinical animal models of chronic cerebral
hypoperfusion studies have repeatedly identified increased inflammation and
oxidative stress. This led to the hypothesis for this thesis; that inflammation and
oxidative stress are key drivers of structural and functional white matter disruption
when cerebral blood flow is reduced.
The studies reported in this thesis were developed to investigate mechanisms
involving inflammation and oxidative stress that can inform future treatments aimed
at preventing the disruption of white matter and cognitive impairment in VCI. One such
mechanism is the Nrf2 (Nuclear factor erythroid 2-related factor 2) signalling pathway.
Nrf2 is a transcription factor that acts to detect and resolve inflammation and oxidative
stress via induction of over 200 antioxidant and anti-inflammatory genes. Studies
have shown that modulation of Nrf2 alters levels of inflammation and oxidative stress
which impact on disease progression in models of Alzheimer’s disease, Parkinson’s
disease and multiple sclerosis. To date, no one has investigated the direct role of Nrf2
in cerebral hypoperfusion-induced white matter disruption. While Nrf2 represents a
promising network approach, another targeted mechanism of interest is microglial
proliferation. Many neurodegenerative diseases including human VCI demonstrate
increases in microglia, a sign of chronic neuroinflammation thought to be detrimental
to cells, tissues and synapses. Work by our group has found an association between
increasing numbers of microglia and the progressive disruption of white matter
structure and function when cerebral blood flow is reduced in a mouse model,
however, whether this is cause or consequence has yet to be determined. The first study of this thesis aimed to test the hypothesis that deficiency of Nrf2
exacerbates white matter pathology and cognitive decline when cerebral blood flow
is reduced. Using wild type and Nrf2 knockout mice the study investigated cortical
perfusion, white matter disruption and gliosis, cognitive impairment and white matter
gene changes following sham or surgically-induced cerebral hypoperfusion (bilateral
carotid artery stenosis). There were no differences in the severity of blood flow
reductions between genotypes initially, however, wild type mice displayed improved
recovery compared to Nrf2 deficient mice. Hypoperfusion induced white matter
disruption and microgliosis in the corpus callosum and the optic tract in both
genotypes, exacerbated by the absence of Nrf2. Further, hypoperfusion induced white
matter astrogliosis and upregulated pro-inflammatory gene signalling in the optic tract
and induced an impairment in spatial working memory. However, these measures
were not affected by Nrf2 deficiency. The results demonstrate that the absence of
Nrf2 exacerbates white matter pathology and microgliosis following cerebral
hypoperfusion but does not impact on functional outcome.
The second study aimed to test the hypothesis that enhancing astrocytic Nrf2-
signalling preserves white matter structure and cognitive decline when cerebral blood
flow is reduced. Astrocytes have larger antioxidant capacity than other cell types in
the brain and overexpressing Nrf2 in astrocytes is associated with reduced white
matter damage in a model of multiple sclerosis, as well as improved outcome in
models of Parkinson’s and Huntington’s disease. Similar to the first study, wild type
mice and mice overexpressing Nrf2 in astrocytes (GFAP-Nrf2) were subjected to
bilateral carotid artery stenosis and cortical perfusion, white matter disruption and
gliosis, cognitive impairment and white matter gene changes were assessed. There
were no differences in the severity of blood flow reductions between genotypes. Akin
to the first study, hypoperfusion induced white matter disruption, micro- and
astrogliosis and pro-inflammatory gene signalling in the optic tract. The majority of
these alterations were ameliorated in GFAP-Nrf2 mice. In addition, the impairment in
spatial working memory induced by cerebral hypoperfusion was modestly improved
in GFAP-Nrf2 mice compared to wild type controls. These findings support the
hypothesis that astrocytic Nrf2 preserves white matter structure and function following
cerebral hypoperfusion.
The first two studies identified structural and functional consequences of altered
inflammation mediated via alterations in Nrf2 signalling. To thoroughly investigate the
Nrf2 signalling pathway following cerebral hypoperfusion the next step would ideally have been to study microglial Nrf2, however due to the lack of a suitable animal
model, the third and final study instead aimed to test the hypothesis that microglial
colony-stimulating factor 1 receptor (CSF1R) signalling is a driver of white matter
disruption and cognitive decline when cerebral blood flow is reduced. Wild type mice
treated with a pharmacological inhibitor of CSF1R (GW2580) or vehicle control, as an
oral gavage or in diet, were studied by a similar experimental protocol as the first two
studies. There were no differences in the severity of cerebral hypoperfusion between
GW2580- or vehicle-treated animals either at one or six weeks following bilateral
carotid artery stenosis. One week of GW2580 treatment was shown to modulate
microglial proliferation and pro-inflammatory signalling in white matter. Remarkably,
treatment with GW2580 for six weeks completely rescued impairments in spatial
learning, protected against white matter disruption and prevented increased both
white matter micro- and astrogliosis compared to wild type controls. These results
suggest that CSF1R signalling in microglia is an important driver of the
pathophysiological mechanisms that lead to white matter disruption and cognitive
impairment when cerebral blood flow is reduced, and importantly, that targeted
inhibition of this improves functional outcome.
In conclusion, the work described in this thesis provides evidence of the
contribution of inflammation and oxidative stress to the disruption and functional
impairment of cerebral white matter. The results indicate that these mechanisms are
amenable to alteration, and that direct microglial inflammatory mechanisms play an
important role in the pathogenesis of white matter disruption and cognitive decline.
The results demonstrate that targeted inhibition of CSF1R signalling in microglia and
increased astrocytic Nrf2 expression leads to improved structural and functional
outcome and as such represent a basis for potential treatment which warrants further
investigation
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