173 research outputs found
Investigation of inflammatory and oxidative stress mechanisms in the disruption of white matter structure and function following chronic cerebral hypoperfusion
No full textVascular 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
JCB887777 Supplementary material - Supplemental material for The mitochondrial calcium uniporter is crucial for the generation of fast cortical network rhythms
No full textSupplemental material, JCB887777 Supplementary material for The mitochondrial calcium uniporter is crucial for the generation of fast cortical network rhythms by Carlos Bas-Orth, Justus Schneider, Andrea Lewen, Jamie McQueen, Kerstin Hasenpusch-Theil, Thomas Theil, Giles E Hardingham, Hilmar Bading and Oliver Kann in Journal of Cerebral Blood Flow & Metabolism</p
Control of anti-apoptotic and antioxidant pathways in neural cells
No full textOxidative 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
TCN 201 selectively blocks GluN2A-containing NMDARs in a GluN1 co-agonist dependent but non-competitive manner
No full textAntagonists that are sufficiently selective to preferentially block GluN2A-containing N-methyl-d-aspartate receptors (NMDARs) over GluN2B-containing NMDARs are few in number. In this study we describe a pharmacological characterization of 3-chloro-4-fluoro-N-[4-[[2-(phenylcarbonyl)hydrazino]carbonyl]benzyl]benzenesulphonamide (TCN 201), a sulphonamide derivative, that was recently identified from a high-throughput screen as a potential GluN2A-selective antagonist. Using two-electrode voltage-clamp (TEVC) recordings of NMDAR currents from Xenopus laevis oocytes expressing either GluN1/GluN2A or GluN1/GluN2B NMDARs we demonstrate the selective antagonism by TCN 201 of GluN2A-containing NMDARs. The degree of inhibition produced by TCN 201 is dependent on the concentration of the GluN1-site co-agonist, glycine (or d-serine), and is independent of the glutamate concentration. This GluN1 agonist-dependency is similar to that observed for a related GluN2A-selective antagonist, N-(cyclohexylmethyl)-2-[{5-[(phenylmethyl)amino]-1,3,4-thiadiazol-2-yl}thio]acetamide (TCN 213). Schild analysis of TCN 201 antagonism indicates that it acts in a non-competitive manner but its equilibrium constant at GluN1/GluN2A NMDARs indicates TCN 201 is around 30-times more potent than TCN 213. In cortical neurones TCN 201 shows only modest antagonism of NMDAR-mediated currents recorded from young (DIV 9-10) neurones where GluN2B expression predominates. In older cultures (DIV 15-18) or in cultures where GluN2A subunits have been over-expressed TCN 201 gives a strong block that is negatively correlated with the degree of block produced by the GluN2B-selective antagonist, ifenprodil. Nevertheless, while TCN 201 is a potent antagonist it must be borne in mind that its ability to block GluN2A-containing NMDARs is dependent on the GluN1-agonist concentration and is limited by its low solubility
There and back again: functional outcomes of reciprocal neuron-astrocyte signalling
No full textNeurons 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
Contribution of the centriolar protein Trichoplein to endothelial cell function in brain vasculature
No full textAge-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
Coupling of the NMDA receptor to neuroprotective and neurodestructive events
No full textNMDA (N-methyl-D-aspartate) receptors are a subtype of ionotropic glutamate receptor with an important role in the physiology and pathophysiology of central neurons. inappropriate levels of Ca2+ influx through the NMDA receptor can contribute to neuronal loss in acute trauma such as ischaemia and traumatic brain injury, as well as certain neurodegenerative diseases such as Huntington's disease. However, normal physiological patterns of NMDA receptor activity can promote neuroprotection against both apoptotic and excitotoxic insults. As a result, NMDA receptor blockade can promote neuronal death outright or render neurons vulnerable to secondary trauma. Thus responses to NMDA receptor activity follow a classical hormetic dose-response curve: both too much and too little can be harmful. There is a growing knowledge of the molecular mechanisms underlying both the neuroprotective and neurodestructive effects of NMCA receptor activity, as well as the factors that determine whether an episode of NMCA receptor activity is harmful or beneficial. it is becoming apparent that oxidative stress plays a role in promoting neuronal death in response to both hyper- and hypo-activity of the NMDA receptor. increased understanding in this field is leading to the discovery of new therapeutic targets and strategies for excitotoxic disorders, as well as a growing appreciation of the harmful consequences of NMDA receptor blockade
Pro-survival signalling from the NMDA receptor
No full textCa2+ influx through the NMDA (N-methyl-D-aspartate) subtype of ionotropic glutamate receptors plays a Jekyll and Hyde role in the mammalian central nervous system. While it mediates excitotoxic death triggered by stroke and other acute trauma, there is growing evidence that physiological levels of NMDA receptor activity promote survival. Understanding the mechanisms that underlie these opposing effects may lead to strategies to selectively block pro-death signalling, which could have considerable clinical benefits
The consequences of neurodegenerative disease on neuron-astrocyte metabolic and redox interactions
No full textBrain metabolic pathways relating to bioenergetic and redox homeostasis are closely linked, and deficits in these pathways are thought to occur in many neurodegenerative diseases. Astrocytes play important roles in both processes, and growing evidence suggests that neuron-astrocyte intercellular signalling ensures brain bioenergetic and redox homeostasis in health. Moreover, alterations to this crosstalk have been observed in the context of neurodegenerative pathology. In this review, we summarise the current understanding of how neuron-astrocyte interactions influence brain metabolism and antioxidant functions in health as well as during neurodegeneration. It is apparent that deleterious and adaptive protective responses alter brain metabolism in disease, and that knowledge of both may illuminate targets for future therapeutic interventions.</p
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