Theses - Clinical Neurosciences

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  • ItemEmbargo
    Relationship of brain functional, structural and vascular patterns to cognition in ageing and dementia
    Liu, Xulin; Liu, Xulin [0000-0002-8219-2848]
    Cognitive decline with neurodegeneration is a major healthcare burden. To tackle this burden, it is necessary to better understand the brain-cognition relationship in ageing and neurodegenerative diseases especially at an earlier stage, ideally before cognitive impairment. Measurements from neuroimaging can be useful to identify the complex processes of neurodegeneration which involve structural, cerebrovascular and functional changes. In this dissertation, I investigated the following questions: 1) Can we explain the relationship between brain architecture and cognition by the integration, or dissociation, of structural connectivity and functional connectivity? 2) Do the patterns of functional network topography, brain structure and cerebral blood flow have independent or synergistic effects on cognitive function in healthy ageing? 3) Do the patterns of functional network topography, brain structure and cerebral blood flow have independent or synergistic effects on cognitive function at the presymptomatic stage of dementia? To address question 1, I investigated whether the increased reliance on maintaining functional connectivity for good cognition in old age is facilitated by structural network connectivity or independent of structural network connectivity. I used multivariate integrative approaches to assess the relationship to cognition across the adult lifespan of (i) shared signals between structural and functional connectivity and (ii) unique signals in functional connectivity that are independent of differences in structural connectivity. I found that the maintenance of cognitive functions in older people depends on functional connectivity supported by strong structural connectivity. This dependency was related to education level. To address questions 2 and 3, I combined multimodal neuroimaging to investigate the differences associated with cognitive decline across the adult lifespan and in genetic frontotemporal dementia. On healthy ageing subjects from the Cam-CAN cohort, I applied linked independent component analysis which allows for simultaneous characterization of MRI-based structural, cerebrovascular, and functional measurements. The purpose was to integrate all of these neuroimaging signals to better understand their contribution to cognitive decline in ageing. I then applied the same method to presymptomatic carriers of genetic mutations associated with frontotemporal dementia using the GENFI cohort. I found that among these brain patterns, functional network integrity, particularly the frontoparietal network integrity, was the most sensitive to the effects of genetic mutations and significantly correlated with cognitive performance both in healthy ageing and presymptomatic frontotemporal dementia mutation carriers. Together the findings suggest that brain structural, cerebrovascular and functional networks show cognition-related alternations while the individuals are still cognitively normal. This process can be affected by age, genetic mutation, education and the interplay between patterns within the brain. Functional network integrity and connectivity are particularly important for the maintenance of normal cognition, especially in older subjects and in those approaching the likely year of dementia onset. Integrating multiple neuroimaging measurements could be a promising approach to stratify phenotypic and genotypic heterogeneity in otherwise-fuzzy cognitive variability in ageing and neurodegenerative diseases.
  • ItemOpen Access
    The role of Toll-like receptors in Parkinson's disease
    Kennedy, Catherine
    Inflammation in the brain has been strongly implicated in the development and progression of Parkinson’s disease (PD). Neuroinflammation in PD is thought to occur in response to aberrant α-synuclein aggregates, and may be mediated by Toll-like receptors (TLRs). TLRs are pattern recognition receptors with the ability to detect damage-associated molecular patterns. The activation of TLRs, potentially occurring through the detection of aggregated α-synuclein, triggers the release of pro-inflammatory cytokines. This may result in chronic inflammation, which produces an environment toxic to neurons and thus causes neurodegeneration such as that seen in the substantia nigra of the PD brain. I hypothesise that the blockade of TLRs will result in the slowing of pathological progression in an animal model, and the blockade of an α-synuclein inflammatory response in human PBMCs. Chapter 1 describes the clinical and pathological characteristics of PD and some of the suggested mechanisms behind the progression of pathology, focusing on neuroinflammation. Additionally, it introduces TLRs and summarises the current literature surrounding their connection to PD, in particular that of TLRs 2 and 4. Chapter 2 describes the reproduction of the Kuan et al., (2019) α-synuclein animal model, including behavioural and neuropathological features. To test the effect of TLR blockade on the development of pathology the drug candesartan was used. This is a licensed drug used in the treatment of hypertension through its action as an AT1 receptor blocker, but has also been identified as decreasing the expression of TLRs 2 and 4. This work demonstrated a protective effect of TLR blockade on the development of cholinergic neurodegeneration and olfactory deficits, both features of early human PD. Chapter 3 investigates the mechanism behind the protective effects of candesartan seen in chapter 2. Candesartan showed only small trends towards decreasing the expression and protein levels of TLRs 2 and 4. Neuroinflammation was not identified within the brain, and thus candesartan did not appear to have any effects on this inflammation. Peripheral inflammation was identified at a 2-month timepoint, and candesartan decreased this inflammation, providing a potential protective mechanism. Chapter 4 describes the optimisation of an *in vitro* assay to investigate the inflammatory response of human peripheral immune cells to different forms of α-synuclein. This work showed that human peripheral immune cells show a pro-inflammatory response to both monomeric and oligomeric α-synuclein, and that this is blocked using candesartan. This response was shown to be TLR4- but not TLR2-dependent. Chapter 5 summarises the main conclusions of my thesis. Candesartan is having a protective effect on the development of neurodegeneration within the α-synuclein animal model, however the mechanisms behind this are unclear. This protective effect could arise through the suppression of peripheral inflammation, through the inhibition of autophagy through decreasing the expression of TLRs on neurons, or through the blockade of AT1 receptors. Further evidence supporting an anti-inflammatory mechanism arises from the decrease in α-synuclein-mediated inflammation in *in vitro* human peripheral immune cells on candesartan treatment. This thesis finishes with suggestions for further work to provide more insight into the mechanisms behind the protective action of candesartan, and to provide further rationale for the use of candesartan in a clinical trial.
  • ItemOpen Access
    Disease mechanisms and markers of progression in cerebral small vessel disease
    Brown, Robin; Brown, Robin [0000-0003-0431-7841]
    Introduction
    Cerebral small vessel disease (SVD) is a common disease process accounting for a quarter of all ischaemic strokes, around 80% of haemorrhagic strokes, and is the major contributor to vascular cognitive impairment and dementia. Despite it being a major public health burden, understanding of the natural history is incomplete and the specific pathophysiological processes involved have not been fully elucidated. Consequently, there are few effective disease modifying treatments. In part I of this thesis I aimed to clarify elements of the natural history of white matter hyperintensity lesions (WMHs) in SVD. These are a key radiological feature of SVD that are strongly correlated with clinical sequelae, and I further tested whether brain lesion volume can regress over time. In part II I investigated the role of two novel pathophysiological mechanisms (inflammation and the permeability of the blood-brain barrier), and their relationship with SVD severity and progression. Methods
    I performed a systematic review of WMH growth and used inverse variance-weighted meta-analysis to determine the expected WMH change over time in high-risk populations. I next used a novel timepoint-blind WMH marking technique to assess whether WMH volume regresses over time in three separate SVD cohorts. Finally I studied a cohort of patients with SVD undergoing PET-MRI imaging, phlebotomy and neuropsychometric testing. Results
    WMHs typically expand at 2.50 ± 3.02 cc/year in patients with SVD and this is significantly more likely in patients with hypertension and who currently smoke. I found only 12/417 participants (2.9%) who showed modest WMH regression on longitudinal imaging, and this was more likely in patients with less severe disease at baseline. I demonstrated significant differences between patient and control groups in both microglial signal and blood-brain barrier permeability, and associations between microglial signal and both clinical and radiological markers of SVD severity. These disease processes did not predict disease progression at one year. Conclusions
    I calculated the expected rate of WMH growth in relevant populations and how these data affect the sample sizes required to show a treatment effect, which should inform future trials. My results investigating WMH regression suggest that this is unlikely to be a significant factor in severe SVD. I showed that both microglial signal and blood-brain barrier permeability are likely to be relevant in SVD, but whether they are disease causing remains unclear. I further discussed the ongoing interventional study in which the data collection for this thesis was nested (MINERVA). The MINERVA trial aims to answer this question by testing whether minocycline can inhibit activated microglia and stabilise the blood-brain barrier, and I presented baseline data from the trial.
  • ItemOpen Access
    Mitochondrial Dysfunction and Stress Responses in CHCHD10 Myopathy and Neurodegeneration
    Shammas, Mario
    In the last decade, dominant mutations in the mitochondrial protein CHCHD10 and its paralogue CHCHD2 were shown to cause familial amyotrophic lateral sclerosis and Parkinson’s disease, respectively, with phenotypes that often resemble the idiopathic forms of the diseases. Different mutations in CHCHD10 cause additional neuromuscular disorders, including the lower motor neuron disease spinal muscular atrophy Jokela type and autosomal dominant isolated mitochondrial myopathy. Modelling these disorders is revealing how mitochondrial dysfunction contributes to the aetiology of neuromuscular and neurodegenerative diseases. In this dissertation I generate and characterise a knockin mouse model of the CHCHD10 p.G58R mutation, which in humans causes a mitochondrial myopathy and cardiomyopathy. The G58R mouse recapitulates the human phenotype, developing a severe myopathy from birth and a cardiomyopathy later in life. I found that mutant CHCHD10 forms aggregates in affected tissues, applying a toxic protein stress to the inner mitochondrial membrane as evidenced by prominent dilations of cristae containing membranous inclusions. Unexpectedly, the survival of CHCHD10-KI mice depends on a protective stress response mediated by the mitochondrial metalloendopeptidase OMA1. The OMA1 stress response acts both locally within mitochondria, causing mitochondrial fragmentation, and signals outside the mitochondria, activating the integrated stress response through cleavage of DAP3-binding cell death enhancer 1 (DELE1). I additionally identify an isoform switch in the terminal complex of the electron transport chain as a component of this response. Furthermore, I perform ultra-high depth sequencing of mitochondrial DNA (mtDNA) of CHCHD10 mutant mice and show that mtDNA deletion levels are higher in affected tissue, and the accumulation of these deletions happens in an age-dependent manner. I finally show that in addition to accelerating the rate of naturally occurring deletions, CHCHD10 mutations also lead to the accumulation of a novel set of deletions characterised by shorter direct repeats flanking the deletion breakpoints. I therefore here demonstrate that CHCHD10 mutations cause protein aggregation, inner membrane instability and mtDNA deletions with a distinct signature. This leads to the activation of an OMA1 stress response which is critical for neonatal survival, coordinating local and global stress responses to reshape the mitochondrial network and proteome.
  • ItemOpen Access
    Imaging Correlates of Heterogeneity in the Syndromes Associated with Frontotemporal Lobar Degeneration
    Whiteside, David; Whiteside, David [0000-0002-5890-9220]
    The syndromes associated with frontotemporal dementia are heterogeneous in their presentation and progression, with variable correlation between clinical phenotype and underlying proteinopathy. Single pathologies are associated with diverse clinical presentations, while the same clinical presentation can be caused by multiple pathological entities. Heterogeneity makes predicting underlying pathology and longitudinal outcomes challenging in clinical practice and in research settings. I propose that a multi-modal imaging approach, including structural and task-free functional magnetic resonance imaging, will provide mechanistic insight into how phenotypic variance arises and improve predictions of disease progression and survival. In this thesis I draw from data for participants recruited at the University of Cambridge and from two multi-site collaborations, the Progressive Supranuclear Palsy Corticobasal Syndrome Multiple System Atrophy Longitudinal Study UK (PROSPECT-M-UK) and the Genetic Frontotemporal Dementia Initiative (GENFI). I describe characteristic differences in markers derived from task-free functional MRI and their relationship to patients’ clinical manifestations. I relate these functional changes to imaging markers of neuronal loss, cell death and synaptic loss. I find that subcortical atrophy from structural MRI relates to cortical functional network disruption, and that synaptic loss measured through [11C]UCB-J positron emission tomography affects behaviour in relation to changes in functional connectivity. I investigate differences in functional connectivity across the disease course. In individuals with familial frontotemporal dementia, time-varying functional network abnormalities predict symptomatic conversion in presymptomatic mutation carriers and future cognitive decline in symptomatic participants. In progressive supranuclear palsy and corticobasal syndrome between-network connectivity explains variability in survival but does not improve predictive accuracy beyond clinical and structural imaging metrics. Imaging-derived biomarkers in frontotemporal lobar degeneration need to be appropriately targeted at components of the neurodegenerative cascade. Task-free functional MRI is an objective and scalable neural marker of clinical syndrome, useful in detecting symptomatic onset and prognostication but limited by small effect sizes, poor signal-to-noise ratio, and moderate reliability. I discuss developments required in image acquisition and analysis to support clinical practice and trials of experimental treatments.
  • ItemOpen Access
    Investigating protrudin’s role in neuroprotection and axon regeneration in the central nervous system
    Love, Fiona; Love, Fiona [0000-0002-7805-5234]
    Protrudin — a transmembrane scaffold protein found in tubular regions of the endoplasmic reticulum (ER) — has previously been shown to strongly promote neuronal survival and axon regeneration after central nervous system (CNS) injury. This is in part due to increased levels of integrins in the distal axon, but this mechanism does not fully account for its beneficial effects. We have investigated protrudin's effects on intracellular transport, morphology, and protein localisation in neurons, and found varied but specific effects on different cellular systems. In particular, protrudin does not have any effect on the transport of late endosomes in CNS neurons — despite evidence for this mechanism in other cell types — due to the absence of key adaptor protein FYCO1 in mature neurons. It also does not have any substantial effect on dendritic spine morphology, so it does not indiscriminately promote cellular outgrowth. On the other hand, protrudin does interact with ER export and associated secretory machinery. Overexpression of an active mutant of protrudin increases the amount of an ER-Golgi intermediate compartment in axon terminals, and affects the transport of Golgi satellite organelles, which we observed even in the distal axon. Our data demonstrates that protrudin provides axons with the machinery for local membrane protein synthesis, which may play a role in neuron survival and regeneration. This work opens up new avenues for future research into adult CNS repair.
  • ItemRestricted
    Rebalancing Neurodegeneration in Ataxia-Telangiectasia
    Zhao, Yiqing
    [Restricted]
  • ItemEmbargo
    Unravelling Spiral Ganglion Neuron Electrophysiology: Heterogeneity, Gene Therapy, and In-Vitro Testing Models
    Sevgili, Ilkem
    Hearing impairment is a prevalent global health challenge, and cochlear implants (CIs) have emerged as a transformative solution for individuals with severe to profound hearing loss. However, the variability in CI outcomes, especially in complex listening situations, underscores the need for further advances in CI technology. One major aspect of improved CI performance is a better understanding of spiral ganglion neurons (SGNs). CIs work by directly and electrically stimulating SGNs, thus their performance depends on healthy and excitable SGN populations. This thesis comprises a multifaceted approach aimed at improving CI performance by studying SGN behaviour at three different levels. Firstly, this thesis investigates the intrinsic electrophysiological properties of SGNs at a single-cell level using a patch-clamp technique. Three distinct SGN classes based on their spike adaptation profiles have been identified and the interspike interval of action potentials in each class has been studied. Additionally, the changes in SGN response time or latencies across varying ranges of stimulation amplitudes have been evaluated. Secondly, an *in vitro* gene therapy approach through the activation of the Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Delta (PIK3CD) gene has been investigated for its potential for SGN regeneration. This gene encodes for phosphoinositide 3-kinase (PI3K), a key initiator of the PI3K/Akt/mTOR pathway. The results of this therapy show enhanced neurite outgrowth and survival of SGNs. Furthermore, regenerated SGNs preserve their electrical activity and even possess enhanced excitability. This research establishes gene therapy as a promising avenue for SGN regeneration, offering potential benefits to CI users reliant on SGN viability and excitability. Finally, this thesis pioneers the development of a cochlea-on-a-chip platform, designed to record population responses from SGNs in response to a real CI. This innovative platform supports SGN survival, replicates cochlear current spread dynamics and holds promise for a deeper understanding of CI-SGN interactions. Collectively, this research helps us better understand SGN electrophysiology, explores SGN regeneration through gene therapy, and proposes a novel cochlea-on-a-chip platform, hereby potentially advancing CI technology and improving auditory experiences for those with hearing impairments.
  • ItemEmbargo
    Chronic activation and downstream mechanisms of programmed axon death
    Antoniou, Christina
    Axon loss is a characteristic feature shared among various neurodegenerative disorders, regardless of their distinct primary causes. Programmed axon death is a conserved, well-characterised pathway of axon degeneration activated by physical injury and in disease states. The two main regulators of the pathway are the pro-survival NAD-synthesising enzyme NMNAT2, and the pro-degenerative NAD(P)-consuming enzyme SARM1. Over-expression of NMNAT enzymatic activity and removal of SARM1 can significantly delay axon degeneration following numerous neurodegenerative stressors in vitro and in several disease models in vivo, including traumatic brain injury, Parkinson’s disease and glaucoma. Association of these components with human disease is becoming increasingly apparent, with mutations in NMNAT2 identified in patients with polyneuropathy and hyperactive SARM1 variants found to be enriched in patients with motor nerve disorders. While the pathway’s role in axon degeneration caused by acute insults has been well characterised, the emerging clinical data highlight the need to study the contribution of programmed axon death to chronic conditions which characterise the majority of neurodegenerative diseases. This thesis has a multifaceted aim, seeking to investigate the possibility of partial, chronic SARM1 activation in morphologically intact axons, establish functional interactions between pathway components, and explore the mechanisms downstream of SARM1 that drive axon degeneration. Using mice with graded levels of NMNAT2 protein, this thesis demonstrates that sub-heterozygous NMNAT2 expression reduces viability in a SARM1-dependent manner and partially activates SARM1 in primary neuronal cultures. Furthermore, the NAD precursor nicotinamide riboside (NR), can decrease NAD levels in neurites expressing low levels of NMNAT2. In an attempt to test for synergy between NMNAT2 and another axon-protective protein, Stathmin-2, it becomes evident that the proteins mediate their effects on axon maintenance through distinct pathways. Regarding mechanisms downstream of SARM1 activation, the data in this thesis support that primary neurons are able to survive with critically low NAD levels, while variations in NADP levels might represent a more appropriate indication of axon survival. Finally, NaADP, a calcium mobiliser produced by SARM1, emerges as a candidate mechanism driving axon degeneration. The findings presented in this thesis support that sub-clinical activation of programmed axon death can occur in conditions not directly associated with axon degeneration and propose mechanisms by which SARM1 could drive the axon degeneration process.
  • ItemOpen Access
    Effect of cochlear shape and size on cochlear implant insertion forces
    Hrncirik, Filip
    This PhD thesis constitutes a comprehensive investigation into the critical factors and parameters influencing cochlear implantation outcomes, with the ultimate aim to enhance surgical practices and patient outcomes. Chapter 2 offers a detailed overview of the anatomical and physical properties of the cochlea to aid the development of accurate models for improved future cochlear implant (CI) treatments. It highlights the advancements in the development of various physical, animal, tissue engineering, and computational models of the cochlea, along with the challenges and potential future directions. Chapter 3 performs a systematic review, consolidating and scrutinising the existing literature on cochlear implantation. Firstly, it centres on the determinants of insertion forces (IFs) and intracochlear pressure (IP) during cochlear implantation, focusing on insertion depth, speed, and the role of robotic assistance. The findings underscore the necessity for standardisation across studies and offer critical insights into the factors influencing IFs and IP during cochlear implantation. The second part of the chapter assesses the influence of surgical approach and cochlear implant type on the occurrence and distribution of cochlear trauma, identifying potential areas for improvement. It indicates the significance of implant design and surgical approach in reducing cochlear trauma and enhancing patient outcomes. Chapter 4 scrutinises the precision and transparency of Stereolithography (SLA) and Digital Light Processing (DLP) 3D printing technologies in creating full cochlea and *scala tympani* models. The most accurate and transparent models were achieved using DLP technology with a 30 μm layer height combined with an acrylic coating. This provides a promising pathway for creating detailed artificial cochlea models for use in cochlear implantation surgery. Chapter 5 presents a systematic investigation of the influence of different geometrical parameters of the *scala tympani* on the cochlear implant insertion force. This was done using accurate 3D-printed models of the *scala tympani* with geometrical alterations. The results indicate that the insertion force is largely unaffected by the overall size, curvature, vertical trajectory, and cross-sectional area once the forces were normalised to an angular insertion depth. This supports the Capstan model of the cochlear implant insertion force which suggests the major factor in assessing insertion force and associated trauma are the friction, the tip stiffness, and the angular insertion depth, rather than the length of the CI inserted. This thesis provides novel insights into the dynamics of cochlear implantation, offers a comprehensive appraisal of the current state of research, provides methodologies to fabricate accurate artificial models, and identifies areas for further investigation. It is anticipated that the findings will guide future research and clinical practice to optimise cochlear implantation outcomes.
  • ItemOpen Access
    The role of the type-I interferon response in the aggregation of tau protein
    Sanford, Sophie Andrea Izumi; Sanford, Sophie [0000-0002-7712-0575]
    Alzheimer’s disease (AD) and other tauopathies are neurodegenerative diseases characterised by the abnormal intracellular inclusions of hyperphosphorylated tau protein in neurons and/or glia. In AD, tau protein aggregation is accompanied by extracellular plaques of β-amyloid (Aβ) protein, protein pathologies which feature alongside prominent neuroinflammation. The innate immune response, the first line of defence against foreign pathogens, is emerging as a major contributor to the development of neurodegenerative disease. IFN-Is are soluble cytokines produced in the innate immune response which induce an antiviral transcriptional state via the regulation of several thousand ISGs. Activation of the IFN-I response is observed in the brain in AD and with age. The role of the IFN-I response in the development of Aβ protein pathology, has been well characterised, identifying Aβ as an agonist of the IFN-I pathway. Currently however, the role of IFN-I signalling in the progression of tau pathology has not been explored, and this forms the focus of this PhD thesis. My over-arching hypothesis is that the IFN-I response contributes to the development of tau pathology. I demonstrate that IFN-I promotes tau pathology in physiological cell systems and in vivo. In primary neural cultures, polyI:C, a synthetic analogue of viral nucleic acids, evoked a potent cytokine response that enhanced the seeded aggregation of tau in an IFN-I dependent manner. IFN-I induced vulnerability to seeded tau aggregation was intrinsic to neurons and could be prevented by pharmacological inhibitors. Finally, aged P301S-tau mice lacking *Ifnar1*, the receptor for IFN-I, had significantly reduced tau pathology compared to P301S-tau mice with intact IFN-I signalling. My results suggest that IFN-I plays a critical role in potentiating tau aggregation, identifying the IFN-I response as a potential therapeutic target in AD and other tauopathies. Chapter 1 describes the background and rationale of this PhD thesis, including an introduction to AD, tauopathies, and innate immunity. It also contains a broad overview of inflammation in AD and the contributions of brain-resident innate immune cells, microglia, before focusing on the current understanding of the type-I IFN response in AD and tauopathies. Finally, I present the hypotheses examined in this thesis. Chapter 2 describes the materials and methods used in this PhD thesis. Chapter 3 describes studies conducted in primary neural cultures and organotypic hippocampal slice cultures to examine the effects of IFN-I signalling in a model of seeded tau aggregation. PolyI:C, an agonist of the IFN-I response, exacerbated the aggregation of tau in an IFN-I dependent manner. It also explores the contribution of glial cells in IFN-I driven seeded tau aggregation. Depletion of microglia and astrocytes showed that IFN-I induced vulnerability was intrinsic to neurons. Chapter 4 describes a neuropathological investigation of tau pathology and neuroinflammation in murine models of tau pathology, including in mice with genetic depletion of IFNAR, the receptor for IFN-I. Examination of the cortical cytokine profile showed that tau pathology in P301S-tau mice was associated with an increase in proinflammatory cytokines relative to WT mice, however this was largely unaffected by the genetic depletion of IFNAR, with the exception of two IFN-I regulated cytokines. Hyperphosphorylated tau pathology, and seed-competent tau species were significantly reduced in the cerebral cortex and brainstem of aged *Ifnar1-/-* P301S-tau mice relative to *Ifnar1+/+* P301S-tau mice. In contrast, microgliosis, astrogliosis, lifespan, and levels of sarcosyl insoluble tau were unchanged between groups. Cortical cytokine profile analysis was then conducted to examine neuroinflammation in a novel murine model of Aβ and tau pathology, in which Aβ, but not tau pathology, was associated with changes in the cytokine profile of *AppNL-G-F x MaptP290S* double KI mice. Chapter 5 describes studies examining the effects of pharmacological inhibitors of the IFN-I response in primary neural cultures and in vivo. Both an α-IFNAR blocking antibody and a JAK1/JAK2 inhibitor, baricitinib, were able to prevent the IFN-I driven increase in seeded tau aggregation in neural cultures. Pilot studies of baricitinib administration to P301S-tau mice showed that whilst intraperitoneal administration was well tolerated, there appeared to be limited effects on ISG expression in the brain and spleen, restricting further interpretation of its effects on tau pathology. Chapter 6 describes the key conclusions of this PhD thesis. This work suggests that there is a critical role for the IFN-I response in tau aggregation both *ex vivo* and *in vivo*, potentially identifying the IFN-I response as a therapeutic target of interest in AD and other tauopathies. Following a general discussion of how the findings relate to the current field, future directions for the work are presented.
  • ItemEmbargo
    Modelling and investigating treatments of childhood inherited mitochondrial diseases in zebrafish.
    Munro, Benjamin; Munro, Benjamin [0000-0003-4506-7092]
    Mitochondrial DNA depletion syndromes (MDDS) are a group of severe, individually rare, clinically heterogeneous disorders that primarily affect children. Affected tissues tend to be those with a high energy demand such as the central nervous system and skeletal muscle. MDDS stem from disruption to the replication and maintenance of mitochondrial DNA (mtDNA), which encodes key components of the mitochondrial respiratory chain needed for oxidative phosphorylation. There are currently no cures for MDDS, only supportive therapies that try to alleviate symptoms. Due to their clinical heterogeneity and diverse pathomechanism, there is currently a lack of suitable *in vivo* models that allow for disease characterisation and studying treatment. In this thesis I aimed to develop new zebrafish disease models to try and recapitulate features of MDDS to then trial a therapy, nucleoside supplementation, which has been shown to have positive effects in other MDDS disease models and in recent patient trials. Here, using CRISPR/Cas9 mutagenesis I developed three new zebrafish models of MDDS. The first, focused on RRM2B, a protein involved in maintaining mitochondrial nucleotide pools, where I show nucleoside supplementation rescues several hallmarks of MDDS including mtDNA depletion, movement defects and elevated lactate. The second two models focus on POLG, the key catalytic protein for mtDNA replication, where I was also able to recapitulate several features of POLG MDDS and show that nucleoside supplementation increase mtDNA copy number in a model with a mutation in the linker region, an area associated with some of the most common forms of POLG MDDS. These results help to ratify nucleoside supplementation as a leading therapy prospect for treating MDDS. Lastly, utilising a CRISPR/Cas9 F0 knockout method, I modelled a novel mitochondrial disease in zebrafish, recapitulating patient disease features, including a neuromuscular transmission defect, helping to strengthen the disease genotype-phenotype correlation and highlight the neuromuscular junction as feature of mitochondrial disease.
  • ItemOpen Access
    Unravelling the Genetics of Cerebral Small Vessel Disease
    Cho, Pok Him; Cho, Pok Him [0000-0002-5034-3234]
    Cerebral small vessel disease (CSVD) is a major cause of stroke and vascular dementia. While CSVD is mostly sporadic (caused by a complex mix of environmental and genetic risk factors), its rare monogenic forms have been increasingly identified. The most common of these, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by NOTCH3 variants, and the second most frequent, CADASIL type 2, by autosomal dominant HTRA1 variants. COL4A1/2 variants can cause small vessel stroke and intracerebral haemorrhage. This thesis investigates the genetics of CSVD in the context of monogenic conditions using data from three cohorts. Firstly, I correlate NOTCH3 mutations with disease severity in a cohort of 485 patients with CADASIL. Secondly, I investigate factors that may influence the penetrance of NOTCH3, HTRA1 and COL4A1/2 variants in 454,756 UK Biobank participants. Thirdly, I perform a case-control comparison of genomes in 13,310 participants of the National Institute for Health Research BioResource Rare Disease (NBR-RD) study to identify likely causative variants for familial CSVD in known and novel genes. I demonstrate that variant position and cardiovascular risk factors are likely to influence the disease severity of patients with CADASIL and UK Biobank subjects with familial CSVD variants. I also identify novel rare variants in several genes that may contribute to the development of CSVD in the NBR-RD study, although their exact relationship to CSVD requires further investigation. My study highlights the need for better-quality data and more robust analytical and experimental methods to identify other as-yet-unidentified genes and factors underlying CSVD.
  • ItemEmbargo
    The role of the immune system in Parkinson’s Disease: from neuropathology to treatment.
    Curle, Annabel
    Parkinson’s Disease (PD) is a progressive neurodegenerative disorder characterised by progressive loss of dopaminergic neurons (DAn) in the substantia nigra. In the first part of this thesis (chapters 3-4) I describe the immune characterisation of a human embryonic stem cell (hESC)-derived dopamine neuron progenitor cell (NPC) ahead of its use in the STEM-PD clinical trial for PD. Several groups have shown that hESC-NPCs can generate mature DAn when grafted into rodent models of PD - providing targeted physiological dopamine replacement and improving motor function. hESCs offer a better source of cells than previously used foetal ventral midbrain (fVM) tissue, which provided proof of principle for the replacement of DAn in PD but was associated with ethical and logistical issues. Work using the hESC-derived NPCs has now matured into early clinical trials (including STEM-PD, a collaboration between the Barker lab, Cambridge, and Parmar lab, Lund), however it remains unclear whether grafts of this type will trigger a host immune response leading to rejection. Immune characterisation was performed comparing the NPCs to the undifferentiated RC17 hESCs and the mature DAn, as well as previously well-tolerated fVM tissue. I observed no significant immune response to the NPCs in several *in vitro* assays (PBMC/T cell co-cultures or monocyte-derived dendritic cell co-culture assays), despite their low expression of MHC-class I (which is up-regulated in response to IFNγ). Instead, NPCs appeared immunosuppressive; reducing T cell proliferation and CD25 expression in vitro. While I was not successful in determining the mechanism of suppression, I found it to be largely contact dependent, direct (not requiring T regulatory cells or antigen presentation cells) and not due to IDO-1/tryptophan metabolism, the production of immunoregulatory adenosine or expression of any of the tested co-inhibitory molecules, as has been reported for other PSC-derived products. Cytokine assays revealed some evidence of a T cell response to co-culture with the different cell types, though definitive results were limited by large variability seen across biological replicates. Transcriptomic analysis of the NPCs through their differentiation from ESC to mature DAn (+/- inflammatory stimuli) and the fVM revealed similarities between the NPCs and fVM confirmed by hierarchical clustering analyses, with both cell types mounting a strong upregulation of MHC-I antigen presentation pathways in response to IFNγ stimulation. Differences in the expression of potentially immunoregulatory and immunogenic molecules across cell type, differentiation stage and baseline/inflammatory conditions were also observed. In the second part of this thesis (chapter 5) I explored the regional inflammatory environment of the PD brain using post-mortem samples. While the immune system has been heavily associated with PD pathogenesis and progression, it is still not well defined, and most studies have focused on the local environment around DAn loss and the substantia nigra. Here, I examined four regions of the brain: the prefrontal cortex (PFC; thought to be relatively unaffected in PD), the amygdala (in which microglial activation has been previously reported), the putamen (the site of transplant for the hESC-NPCs in PD cell therapy and more broadly the site innervated by the A9 dopamine neurones typically lost in PD), and the substantia nigra (SN; the major region of neurodegeneration in PD). Using two Nanostring nCounter panels for neuroinflammation and glial profiling (totalling ~1300 genes), I found regional differences in the immune signatures of the PD brain compared to controls, with the strongest inflammatory signature seen in the amygdala and evidence of microglial activation in the putamen, which has not been previously reported. Gene expression and pathway analysis suggested a switch from homeostatic, protective microglia to activated, pro-inflammatory microglia. The PFC and SN showed little to no immune signatures, and instead showed upregulation of genes associated with cell stress, damage, and hypoxia. While further exploration is required (and will be performed throughout my planned post-doctoral projects), these data confirm evidence of regional pathology and immune involvement in PD.
  • ItemEmbargo
    Genotype and phenotype in mitochondrial disorders
    Schon, Katherine; Schon, Katherine [0000-0001-8054-8954]
    Mitochondrial disorders are inherited metabolic conditions caused by pathogenic variants in genes affecting oxidative phosphorylation and ATP synthesis. They are a heterogeneous group of disorders with an incidence of 1 in 5000. They can affect a single organ or multiple systems, with age of onset from prenatal to old age. Organs and systems with high energy requirements such as the brain, peripheral nerves, muscles, heart, eyes, and ears tend to be affected. Mitochondrial disorders can be caused by pathogenic variants in the mitochondrial DNA (mtDNA), which is solely maternally inherited, or by pathogenic variants in more than 300 nuclear genes and can follow any inheritance pattern. mtDNA variants can be homoplasmic (present in all copies of the mtDNA) or heteroplasmic (present in a proportion of the mtDNA). Diagnosis
    Mitochondrial disorders are difficult to diagnose because they are individually rare, and the clinical features overlap with other common or rare disorders. Whole genome sequencing (WGS) offers a promising approach. We analysed 319 families with suspected mitochondrial disorders who were undiagnosed following standard NHS testing and had short read WGS through the 100,000 Genomes Project. Nuclear variants were prioritised based on gene panels, ClinVar pathogenic/likely pathogenic variants and the top ten prioritised variants from Exomiser. mtDNA variants were called using an in-house pipeline and compared to a list of pathogenic variants. Copy number variants and short tandem repeats for 13 neurological disorders were also analysed. Likely causative variants were classified using American College of Medical Genetics (ACMG) guidelines. We found a definite or probable diagnosis in 98/139 (31%). Interestingly, ~60% of diagnoses were non-mitochondrial disorders including developmental disorders with intellectual disability, epileptic encephalopathies, myopathies, other metabolic disorders and leukodystrophies. These would have been missed if a targeted approach was taken, and some have specific treatments. Whole genome sequencing projects also offer an opportunity to study nuclear mitochondrial DNA segments (NUMTs) in much greater detail than previously possible. NUMTs are copies of all or part of the mtDNA inserted in the nuclear genome. Wei Wei developed a pipeline for identifying NUMTs from short read WGS using split reads and junction reads. We investigated whether NUMT insertion is a cause of rare disease or cancer in the 100,000 genomes project, using gene lists from PanelApp and the COSMIC database. We did not find evidence of rare disorders caused by germline NUMT insertion, but we did find a probably causative somatic insertion in a myxoid liposarcoma. Natural history
    Natural history studies tend to be small and recruit participants from specialist clinics. Working together with the National Congenital Anomalies and Rare Diseases Registration Service (NCARDRS), we have established a national registry of individuals with genetically confirmed mitochondrial disorders. The registry contains 1134 affected individuals resident in England (48% male, 15% children). 73% have mtDNA diagnoses, most commonly m.3243A>G, Leber Hereditary Optic Neuropathy, and large scale mtDNA rearrangements. The commonest nuclear genetic diagnoses were SPG7, dominant optic atrophy (OPA1) and autosomal recessive POLG. We have linked to Hospital Episode Statistics, death certificates and the cancer registry. We present survival analysis, causes of death and cancer standardised incidence ratios. Treatment
    Mitochondrial disorders can cause early death or lifelong disability and currently have no cure. One approach to treatment is to induce mitochondrial biogenesis. Nicotinamide Riboside (NR) is an NAD+ booster which showed promising results in two mouse models of mitochondrial disorders. We describe an open label study to test the effect of NR for four weeks in eight individuals with m.3243A>G, m.3243A>T or large scale mtDNA deletions. NR was safe and well tolerated. There was no improvement in six-minute walk test distance, grip strength, SF-36 questionnaire results or phosphocreatine recovery time after exercise on 31P-MRS. There was a marginally significant improvement in the timed up and go test. Muscle biopsy results showed that the citrate synthase and mtDNA copy number (which are measures of mitochondrial biogenesis) were not significantly different after 4 weeks of NR but increased expression was observed for 16 mitochondrial genes. We have explored genotypes and phenotypes in mitochondrial disorders considering diagnosis, natural history, and a possible treatment.
  • ItemEmbargo
    A biohybrid bridge between brain and machine: Development and application of a neural implant using iPSC-derived neuronal membranes
    Kawan, Malak
    Despite the numerous advancements in electronics interfacing with the nervous system over the decades, a long-standing challenge has been to achieve long-term stability *in vivo*. The foreign body reaction (FBR), an internal inflammatory mechanism triggered upon implantation of foreign objects, is responsible for fibrotic encapsulation of implanted neural probes, leading to a gradual decline in device functionality due to the physical barrier it creates between electrodes and host tissue (at the tissue-electrode interface). However, the field has progressed from using rigid electronics to incorporating soft biomaterials that conform to the shape of our soft biology. This thesis aimed to explore a novel solution to FBR in neural implants, proposing the hypothesis that the use of material derived from human-derived neurons themselves as a biological mediator, incorporated in the device design, would result in reduced inflammation and improved therapeutic efficacy and stability *in vivo*. To test this hypothesis, the project fused flexible electronics and bioengineering to extract and form human iPSC-derived neuronal membranes, which were then characterised and studied for their performance and electrophysical sealing properties *in vitro*. The study found that these neuronal membranes displayed high mobility and stability *in vitro* and had improved sealing properties compared to other native membranes. Furthermore, the study revealed that implants integrating neuronal membranes resulted in a reduction of inflammation 28 days post-implantation compared to those without, as confirmed by immunohistochemical analysis and successful electrophysiological recordings. These results hold tremendous potential for the future of biocompatible neural interfaces and chronic therapeutic interventions, as they suggest the possibility of enhancing the signal-to-noise ratio and reducing impedance levels through meticulous manipulation and control of lipid and protein composition.
  • ItemOpen Access
    Characterising In Vivo Brain Function Following Mild Traumatic Brain Injury
    Kelleher-Unger, Isaac
    Traumatic brain injury (TBI) is the most complex disease in the most complex organ and a public health emergency. Sometimes called a silent epidemic, there are at least 50 million TBIs each year globally. This incidence translates to half the world’s population having at least one TBI in their lifetime, and evidence suggests that this number is increasing. At least 70% of TBIs are Mild traumatic brain injuries (mTBIs), yet, despite this incidence, these patients remain understudied on the assumption that they make near-complete recoveries with little to no mortality. However, literature shows that 80% of patients report at least one post-traumatic symptom one-year after injury. Ultimately, research into mTBI aims to improve outcomes for this group of patients — the principal and necessary next steps toward achieving this aim are to characterise mTBI more thoroughly and make diagnosis/prognosis more precise. Notably, how the brain functions — in terms of the connections it makes and the way it processes information — after mTBI is yet to be fully understood. Hence, in this thesis, I aimed to answer two questions: first, can Functional magnetic resonance imaging (fMRI), as an in vivo functional imaging technique, help to more thoroughly characterise brain function after an injury? And, second: can in vivo functional imaging make mTBI diagnosis more precise? I adopted four approaches to answer these questions. In my first chapter, I highlight that there are no macroscopic structural alterations following mTBI. However, individuals with brain injuries showed elevated markers of neuronal/glial damage. These two observations underscore that the absence of macroscopic structural pathology is not evidence of a lack of injury, and highlight the need to characterise mTBI beyond current conventions. In particular, in the absence of macroscopic structural injury, mTBI might be better understood in the functional domain. To this end, I first characterised the functional phenotype of the brain following injury. Here, I quantified global connectivity and global complexity of the brain and found that following injury, the brain becomes hyperconnected and hypocomplex. Moreover, these two measures negatively correlated with each other. Next, I characterised the dynamics of brain connectivity using hidden Markov modelling. Here I found a shift in dynamics: patients with mTBI spend less time in a state dominated by connections between subcortical-cortical regions, shifting to a state characterised by cortico-cortical connections. To establish the importance of this, I used machine learning techniques to investigate if the shift in brain dynamics could predict long-term symptoms, finding that altered cerebral dynamics could achieve a reasonable level of accuracy in predicting long-term symptoms following mTBI . Finally, I investigated how specific functional connections are affected in mTBI. Particularly, given the evidence that sulci are a concentrated location of pathology following mTBI, I investigated how an injury affects sulcal functional connectivity. In this analysis, I found altered interhemispheric sulcal connectivity following injury. However, sulcal and gyral functional connectivity achieved similar accuracies for classifying patients with mTBI from healthy controls, supporting the idea that the effects of mTBI are not localised, but distributed. More importantly, using a novel explanatory artificial intelligence methodology, I showed how sulcal connectivity could personalise mTBI diagnosis in a proof-of-concept manner. In answering my first research question, my results have shown that macroscopic function can help characterise the injured brain, advocating that no TBI is mild and that conceptualising mTBI as a disorder of functional connections can offer new insights. In answering my second research question, my results show evidence that mTBI is a heterogeneous disease. By highlighting that different connections are important for different individuals, I have shown that mTBI needs precision diagnostics and provided a starting point for future research to continue this pursuit.
  • ItemOpen Access
    Long-term Follow-up Following Traumatic Brain Injury: Contextual Understanding and Technology-led Solutions
    Smith, Brandon; Smith, Brandon [0000-0001-8471-1368]
    Traumatic brain injury (TBI) is a global health concern and remains a prominent cause of death and disability. Collecting outcome data at follow-up is crucial for both the clinical management of patients, and for wider research efforts. This data helps to decrease the number of deaths and disabilities caused by injuries worldwide. However, outcome data are generally not reliably or consistently recorded in low- and middle-income countries (LMICs), where 85% of the world’s population lives. Thus, the full picture of outcomes post-TBI in these settings remains largely unknown. This thesis was conducted under the National Institute for Health Research Global Health Research Group on Neurotrauma, a multi-disciplinary global group focused on improving care for those with traumatic brain injury. In this respect, this thesis addresses two key questions: what is the state of play of current long-term follow-up and outcome data collection practices following TBI in LMIC settings? And: What roles might technology-led solutions have in enhancing existing, or establishing new, follow-up and outcome data collection efforts? This thesis employed an exploratory mixed methods design, comprised of three core projects. These projects were encapsulated in the ‘Double Diamond’ design process model, which sought to provide an additional, pragmatic structure underpinning the schedule of works. First, a reflexive thematic analysis of semi-structured interviews was conducted with 18 LMIC neurosurgeons to investigate the current state of play of long-term TBI follow-up in LMICs, the contextual challenges associated with it, and potential technology-led solutions to improve the delivery of services. This led to the second project, a comprehensive scoping exercise to map the current evidence base for follow-up technologies used globally in the monitoring of outcomes following TBI. The third and final project was informed by the findings of the earlier studies, which led to the design, development, and evaluation of a novel, SMS-based follow-up technology (FUT) implemented through a local service evaluation project. Qualitative findings derived from project one identified the many different stakeholders with distinct vested interests in long-term follow-up following TBI. The study also showed that clinical case-volume and patient-facing barriers of geography and poverty act as key agents in LMIC follow-up attrition. Findings from the scoping review identified FUTs, particularly those built upon “common denominator” technologies such as text-messaging and telephony, as feasible ways of maintaining remote contact with and clinical oversight of patients post-discharge. The service evaluation project, which piloted a novel FUT in a high-income country (HIC) setting, proved efficacious, demonstrating good concurrent validity between the traditional, clinician-delivered Extended Glasgow Outcome Scale (GOS-E) and a short-form equivalent delivered autonomously through SMS. In conclusion, long-term follow-up for TBI remains a multifaceted challenge. Though currently in their infancy, novel technologies may have an increasing role in bridging communication gaps between traditional outpatient appointments in the future. Such novel technologies offer a new medium to both assess and monitor patient status, independence, and societal reintegration following injury. Such surveillance may alleviate clinical burden through service transformation and improve patient outcomes. However, in all cases where there are technological solutions to care delivery, due diligence must be exercised so that gaps in service access are bridged rather than further exacerbated.
  • ItemOpen Access
    Modelling brain tumours with network neurosciences and deep learning
    Wei, Yiran; Wei, Yiran [0000-0003-1791-6050]
    Glioma is one of the most common and lethal brain tumours. The clinical diagnosis and prognosis of glioma are challenging due to its heterogeneous nature. In the past, glioma was considered a focal disease with local disruption to the brain. However, recent studies suggested that tumour cells would migrate into normal-appearing brain regions through white matter tracts, implying the widespread impact of gliomas. Meanwhile, quantitative MRI technology, including functional MRI and diffusion MRI, assisted in developing an emerging field called network neurosciences. The network neurosciences model the brain as a network consisting of macro-scale regional connectivity of the brain instead of the micro-scale networks of neurons. The novel brain network approach has the potential to characterise the global impact of brain tumours. This thesis aims to invoke specialised network neuroscience approaches for characterising brain tumours and systematically analyse the global tumour disruption across the brain. First, I provided extensive reviews of brain tumours and network neurosciences. Then I discussed the limitations and current research gaps in the cross-disciplinary field of brain networks of brain tumours. The main finding implied that although there was potential for studying brain networks in brain tumours, the tumour disruption could hinder the robust application of the traditional methodology on brain tumour patients. In Chapter 2, I developed a novel approach for constructing structural brain networks with diffusion MRI. This approach tackled the challenge of constructing brain networks using traditional tractography and brain atlas. For evaluating the proposed method, functional networks were generated as the evaluation baseline of the method. Results showed that brain networks generated from the proposed method had a stronger correlation with the functional networks compared to traditional methods, suggesting the robustness of the new approach. In Chapter 3, I extensively analysed the disruption of brain networks in glioblastoma patients based on the methodology of Chapter 2. To address the heterogeneous disruption of glioblastomas, I classified the regions of brain networks into different categories according to their locations. In particular, the disruption in distantly connected regions was significantly associated with patient performance, cognition, and prognosis, which validated the clinical value of brain networks for glioblastomas and implied the early signs of tumour invasion into the normal-appearing brain. In Chapter 4, to investigate the potential of early tumour invasion outside the tumour regions and tackle the unavailability of diffusion MRI, I developed a graph-based deep-learning model for estimating brain networks from anatomical MRI (e.g. T1, T2). Notably, the model was informed by the prior anatomical knowledge of white matter connectivity. The model successfully predicted IDH mutation status with higher performance than convolutional neural networks, which suggested the feasibility of estimating brain networks in retrospective datasets with only anatomical MRI. In Chapter 5, I combined the prior knowledge of brain network disruption and the clinical significance of tumour geometrics to develop a multi-modal deep learning framework that learnt from brain networks, tumour image, and tumour geometrics simultaneously. The model was built on the hypothesis that multi-modal data respectively represented different views of the aggressiveness of the same tumour. Results showed that this model outperformed other state-of-the-art models in predicting the IDH genotype of glioma. Additionally, visual interpretation suggested the interrelation among multi-modal data. In conclusion, this thesis presented feasible tools and experiments for applying network neurosciences for brain tumour patients, which revealed the potential of the quantified characterisation of tumour impact across the whole brain.