Promoting and measuring remyelination and neuroprotection in clinical trials of people with multiple sclerosis

The most tractable strategy to delay or prevent the progressive phase of MS is to promote endogenous remyelination; doing so restores nerve conduction and prevents demyelinated axons from degenerating. The rate-limiting stage in this process is differentiation of oligodendrocyte progenitor cells (OPCs) into mature, myelinating, oligodendrocytes. In animals, agonism of the retinoid X receptor (RXR)-γ enhances remyelination in this way. Metformin also promotes remyelination, this time by overcoming an age-associated block to the responsiveness of OPCs to pro-differentiation factors. In this PhD I show that bexarotene, a non-selective agonist of the RXR receptor, promotes remyelination in people with relapsing remitting multiple sclerosis. Converging evidence from electrophysiology and neuroimaging in a phase II clinical trial (CCMR One) demonstrate that this occurs in demyelinated lesions and is greatest in lesions located in grey matter regions of the brain. I additionally conducted analyses which suggest an age-dependency of bexarotene’s remyelinating effect and led a follow-up sub study of the trial participants which showed the treatment effect afforded by bexarotene is sustained, years after treatment. Unfortunately, bexarotene was poorly tolerated, and so the legacy of this trial will likely be one of shaping the framework for future assessments of remyelinating therapies. As such, I designed, obtained funding and secured approvals for a clinical trial to test the remyelinating effect of the combination of metformin with clemastine (CCMR Two), implementing lessons from CCMR One in the trial design; this is due to commence participant recruitment in 2021. In exploring treatments for progressive forms of MS, not limited to the process of remyelination, I worked with the MS Society to develop and implement a rigorous, expert-led, evidence-based approach to the selection of licensed drugs for repurposing and testing in clinical trials of people with progressive MS. I reviewed the preclinical and clinical literature for a list of compounds and condensed these into a database of summary documents. These were presented to a panel of experts and people affected by MS, ultimately leading to four treatments being recommended for immediate testing in progressive MS trials: R-α-lipoic acid, metformin, the combination treatment of R-α-lipoic acid and metformin, and niacin. Regrettably, much of my research has been halted by the COVID-19 pandemic. In this PhD I had additionally sought to evaluate electrophysiological techniques for quantifying remyelination and neuroprotection – multifocal VEP and saccadometry – in cohorts of people with MS and in the setting of the CCMR Two trial. Instead, when these studies were delayed, I embraced an opportunity to help the Cambridge COVID-19 research effort, working on the RECOVERY trial in the first wave, and leading my own project to analyse enrolment to treatment trials, ultimately describing the barriers to, and implications of, low recruitment rates in advance of the subsequent waves. Consequently, further exploratory research with electrophysiology and the CCMR Two trial will be the subject of post-doctoral research.Multiple Sclerosis Society of the United Kingdo

Inflammatory Cascades in the Pathogenesis of Multiple Sclerosis Lesions

Multiple sclerosis (MS) is a disease of the human central nervous system (CNS) characterised by inflammation and demyelination. Initially the MS lesion has a distinct histopathological picture with myelin-positive microglia in the midst of apparently intact myelin but minimal perivascular inflammation. Inflammatory mediators produced by these activated microglia may precipitate the infiltration of mononuclear cells and the overt myelin loss seen in actively demyelinating MS lesions. Nuclear factor-κB (NF-κB) is a transcriptional regulator of proteolytic enzymes, adhesion molecules and inflammatory cytokines which rapidly translates extracellular signals into protein synthesis. The immunocytochemical detection of the transcriptionally active form of NF-κB, but not the inhibitory protein IκBα, in the nuclei of microglia in normal human CNS white matter indicates the capability of microglia to respond rapidly to pathological stimuli in the CNS. Activation of NF-κB in MS plaques, evident from the nuclear localisation of the NF-κB subunits RelA, c-Rel and p50 in macrophages, may propagate inflammatory demyelination through upregulation of NF-κB-controlled macrophage genes for inflammatory mediators. In demyelinating disease the plasmin-matrix metalloprotease (MMP) enzymatic cascade promotes blood-brain barrier (BBB) damage, generation of encephalitogenic myelin peptides and activation of pro-inflammatory cytokines. Constitutive expression of MMPs 1, 2, 3 and 9 in glial cells in normal control white matter was demonstrated by immunocytochemistry. However, the lack of tissue (t-PA) and urokinase (u-PA) plasminogen activators in glial cells and the absence of caseinolytic activity as shown by in situ zymography emphasises the latent nature of the plasmin-MMP cascade in normal CNS tissue. In contrast, the co-localisation of t-PA and u-PA, rate-limiting serine-proteases, and MMPs in macrophages and astrocytes in active MS lesions forms the basis of a functional enzymatic cascade. Furthermore, increased amounts and activity of u-PA and MMP-9 in homogenates of active MS plaques coupled with the presence of caseinolytic activity in foamy macrophages implicates these cells as the major source of MMPs, which cause proteolytic damage in MS. Insulin-like growth factors (IGFs) play an important role in development and myelination in the CNS but can also stimulate phagocytosis and production of inflammatory mediators by macrophages. In active MS lesions binding of IGF-II to the IGF receptor on foamy macrophages may induce mitogenic responses and invasiveness of macrophages which can be further enhanced by MMP-mediated proteolytic removal of inhibitory IGF-binding proteins. Similarly, the potent mitogens IGF-I and insulin may stimulate astrocytosis and gliosis. In contrast, oligodendrocytes in normal-appearing white matter do not express IGFs or IGF-I receptor which implies that the oligodendrocyte response to these remyelinating growth factors is impaired. Therefore, the prevailing role of IGFs in MS lesions may be in line with pro-inflammatory mediators promoting macrophage and astrocyte responses to tissue damage. In conclusion, NF-κB activation in microglia and macrophages upregulates the production of PAs and inflammatory cytokines which trigger the plasmin-MMP cascade, leading to BBB damage and enhanced inflammatory cell migration and demyelination in white matter. Influx of IGFs through the damaged BBB and their increased local production may promote myelin phagocytosis and reactive astrocytosis. In turn IGF-mediated upregulation of PAs in glial cells could provide a feedback amplification of the MMP cascade. Therefore, the findings from these studies bring together three systems of mediators, NF-κB, MMPS and IGFs, into a hypothetical model for the propagation of demyelination in MS lesions

Investigation of pathophysiological mechanisms in clinically isolated syndrome using advanced imaging techniques

This thesis concerns an observational study of patients recruited after their first episode of neurological symptoms suggestive of demyelination in the central nervous system and diagnosed either with clinically isolated syndrome or relapsing-remitting multiple sclerosis. In multiple sclerosis, brain tissues can exhibit extensive neuroaxonal microstructural and metabolic abnormalities, but little is known about their presence and significance at the time of the first demyelinating episode. I used a novel multi-parametric quantitative MRI approach, combining neurite orientation dispersion and density imaging (NODDI), which gives information about tissue microstructure, and 23Na MRI, which estimates total sodium concentration, a marker of metabolic dysfunction, in the brains of clinically isolated syndrome patients. I found microstructural and sodium homeostasis alterations in cortical areas of patients that showed clinical relevance. Within the diffuse axonal dispersion found in the normal-appearing white matter, the corpus callosum shared with lesions, signs of axonal damage and metabolic dysfunction, thus emerging as a possible target for early neuroprotective interventions. Structural cortical networks (SCNs) represent patterns of coordinated morphological modifications in cortical areas and they have shown pathophysiological changes in many brain disorders, including multiple sclerosis. I investigated alterations of SCNs at the individual level in this early cohort. Patients showed altered small-world topology, an efficient network organization combining dense local clustering with relatively few long-distance connections. These disruptions were worse for patients with higher lesion load and worse cognitive processing speed indicating that pathophysiological changes in the cortical morphology can influence clinical outcomes. Finally, I hypothesised that the patients in the cohort presenting with optic neuritis may have disturbances in neuropsychological functions related to visual processes. I found that cognitive visuospatial processing is affected after unilateral optic neuritis and improves over time with visual recovery, independently of the structural damage in the visual and central nervous system

In vivo imaging of blood-brain barrier disruption in a multiple sclerosis animal model

La sclérose en plaques est une maladie inflammatoire du système nerveux central qui touche beaucoup de jeunes adultes dans le monde entier. Malgré les efforts de recherche, la cause demeure inconnue. Afin de développer de meilleurs traitements et de trouver un remède, les modèles animaux sont utilisés pour identifier des biomarqueurs cellulaires. La caractérisation de ces modèles est essentielle afin de bien transposer les résultats à la maladie humaine et de tester de nouveaux médicaments. Cette thèse est le résultat d'un projet de maîtrise au Centre de recherche de l'Institut universitaire en santé mentale de Québec (CRIUSMQ) qui visait à étudier la barrière hématoencéphalique en tant que biomarqueur possible chez un modèle animal de la sclérose en plaques. Le chapitre 1 présente les différents thèmes abordés dans ce projet. Les chapitres 2, 3 et 4 se concentrent sur les méthodes choisies, tandis que le chapitre 5 présente les résultats obtenus.Multiple sclerosis is an inflammatory disease of the central nervous system that affects many young adults worldwide. Despite research efforts, the cause of the disease remains unknown. In order to develop better therapies and ultimately find a cure, animal models are used to identify cellular biomarkers. Characterizing these models is essential in order to properly relate findings to the human disease and test possible medications. This thesis is the result of a three-year Master's project at Centre de recherche de l'Institut universitaire en santé mentale de Québec (CRIUSMQ) that aimed to study the blood-brain barrier as a possible biomarker of disease pathology in a multiple sclerosis animal model. Chapter 1 introduces the different themes addressed in this project. Chapters 2, 3 and 4 focus on the materials and methods chosen, while chapter 5 presents the results obtained. These findings are discussed in chapter 6

A study of classical and novel markers of disease in multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory and degenerative condition of the Central Nervous System. Focal demyelinating lesions are its neuropathological hallmark, but widespread abnormalities found in otherwise “normal-appearing” tissue are better associated with disability outcomes. HMGB1 is a promiscuous sensor of cellular stress, acting as a link between sterile damage and innate immune mechanisms, with its extra-nuclear release producing diverse outcomes. We report novel findings of significantly increased HMGB1 expression throughout the brain tissue of MS vs. non-MS patients, particularly in macrophages/microglia and oligodendrocytes (OGD). In addition, cerebrospinal fluid HMGB1 levels were increased in early-stage MS patients compared to non-inflammatory control patients. HMGB1 stimulation in-vitro upregulates expression of its receptors in an OGD cell line, potentially propagating chronic inflammation. Expression of the Leucine Rich Repeat and Ig-domain-containing molecules, AMIGO-3 and LINGO-1 is also significantly increased by HMGB1 stimulation in-vitro. These molecules demonstrate particularly intense immunoreactivity in human brain tissue taken at biopsy, at an early disease stage. Thus, exogenous HMGB1 may influence neurodegenerative processes via AMIGO-3 and LINGO-1 and blocking their function could have therapeutic value. Increased expression of HMGB1 in OGD, however, may highlight endogenous neuroprotective mechanisms in response to an unknown trigger

Pathology of the spinal cord in progressive multiple sclerosis (primary progressive vs secondary progressive)

Background: Recent studies have shown that the two major forms of multiple sclerosis are different in the degree of demyelination and atrophy, degree of inflammation, and extent of axonal loss. However, the majority of the previous studies that compared primary progressive and secondary progressive multiple sclerosis were carried out at the brain level. Material and methods: Human post-mortem spinal cords were used to compare the two progressive subtypes. In this project, the 5 major pathological changes associated with MS were studied in the spinal cords of primary progressive and secondary progressive multiple sclerosis. These changes include degree of demyelination, atrophy of the tissue, oligodendrocytes pathology, axonal loss, and neuronal pathology. Results: There was significant atrophy in the spinal cords of MS compared to healthy controls, which affects mainly the upper cord levels. There is a greater degree of demyelination and atrophy affecting secondary progressive compared to primary progressive especially in the upper cord levels. Oligodendrocytes numbers are dramatically reduced in the chronic lesions of WM and GM lesions. But there was high numbers of oligodendrocytes in the normally appearing GM of secondary progressive multiple sclerosis. There was greater reduction in axonal density in the secondary progressive sample especially in the normally appearing WM. Neurons were reduced in the demyelinated grey matter regions with no difference between the two disease forms in this respect. Conclusions: SPMS seem to have greater degree of tissue destruction in the form of demyelination, atrophy, and axonal loss in the normally appearing WM. However, SPMS showed greater numbers of oligodendrocytes in the demyelinated areas of the WM and the GM. Although the disability scale in the two examined groups was found to be similar, the tissue damage appeared to be variable

Pathology of the spinal cord in progressive multiple sclerosis (primary progressive vs secondary progressive)

Background: Recent studies have shown that the two major forms of multiple sclerosis are different in the degree of demyelination and atrophy, degree of inflammation, and extent of axonal loss. However, the majority of the previous studies that compared primary progressive and secondary progressive multiple sclerosis were carried out at the brain level. Material and methods: Human post-mortem spinal cords were used to compare the two progressive subtypes. In this project, the 5 major pathological changes associated with MS were studied in the spinal cords of primary progressive and secondary progressive multiple sclerosis. These changes include degree of demyelination, atrophy of the tissue, oligodendrocytes pathology, axonal loss, and neuronal pathology. Results: There was significant atrophy in the spinal cords of MS compared to healthy controls, which affects mainly the upper cord levels. There is a greater degree of demyelination and atrophy affecting secondary progressive compared to primary progressive especially in the upper cord levels. Oligodendrocytes numbers are dramatically reduced in the chronic lesions of WM and GM lesions. But there was high numbers of oligodendrocytes in the normally appearing GM of secondary progressive multiple sclerosis. There was greater reduction in axonal density in the secondary progressive sample especially in the normally appearing WM. Neurons were reduced in the demyelinated grey matter regions with no difference between the two disease forms in this respect. Conclusions: SPMS seem to have greater degree of tissue destruction in the form of demyelination, atrophy, and axonal loss in the normally appearing WM. However, SPMS showed greater numbers of oligodendrocytes in the demyelinated areas of the WM and the GM. Although the disability scale in the two examined groups was found to be similar, the tissue damage appeared to be variable

ROLE OF EBV IN NEUROINFLAMMATION: IMPLICATIONS FOR MULTIPLE SCLEROSIS PATHOGENESIS

Multiple sclerosis (MS) is a demyelinating disease of the CNS with unknown cause. Individuals who are genetically predisposed and exposed to specific environmental factors have an increased risk of developing MS. Epstein-Barr virus (EBV) infection is linked to MS development, according to a large body of seroepidemiological and pathological evidence. EBV is a ubiquitous human herpesvirus that typically produces silent infection but can also cause a wide range of illnesses. A large cohort of MS and non-MS control cases has been previously examined and revealed the prevalent presence of EBV in MS. However, the role of the virus in the disease is unclear. The main objective of this research was to understand the viral dynamics in vivo and the consequences of peripheral infection on the CNS. To this end, a novel rabbit model of EBV, which produces latent infection comparable to the persistent infection in human carriers, was used in this study. The present dissertation contains (1) human study and (2) animal study for correlation of the results. In the human study, EBV-positive MS cases were examined for histopathological changes. In the animal study, EBV was injected intravenously in one group of animals, and PBS was injected in the control group, with and without immunosuppression. Histopathological changes and viral dynamics were evaluated in the peripheral blood, spleen, brain, and spinal cord, using molecular and histopathology techniques. A number of important aspects of EBV infection were revealed. Peripheral EBV infection led to CNS infection and promoted neuroinflammation in the form of immune aggregates. EBV infected B cells were most likely the source of CNS infection. The immune aggregates were more prevalent in immunosuppressed animals and consisted of focal accumulation of macrophages surrounded by reactive astrocytes and dispersed B and T lymphocytes. The center of aggregates exhibited signs of myelin destruction. Moreover, studying EBV infection over time revealed that the peak in viral load in the periphery and CNS corresponded to an increase in the occurrence of cellular aggregates in the brain. Additionally, altered expression of viral latent transcripts correlated with upregulation of several proinflammatory cytokines in the periphery and the CNS. Increased expression of IL- 6 at the mRNA and protein level in the brain was associated with neuroinflammation. Finally, several similarities and differences were observed between the pathology in EBV positive MS cases and EBV infected rabbit CNS. Several cellular key players contributed to both pathologies, however, the extent of infiltration of these cells and their distribution differed between the two. This work establishes the first direct in vivo evidence for the role of peripheral EBV infection in CNS pathology, and demonstrates the utility of a novel model for dissecting viral mechanisms involved in the development of EBV- associated diseases including MS

Image Texture Characterization Using the Discrete Orthonormal S-Transform

We present a new efficient approach for characterizing image texture based on a recently published discrete, orthonormal space-frequency transform known as the DOST. We develop a frequency-domain implementation of the DOST in two dimensions for the case of dyadic frequency sampling. Then, we describe a rapid and efficient approach to obtain local spatial frequency information for an image and show that this information can be used to characterize the horizontal and vertical frequency patterns in synthetic images. Finally, we demonstrate that DOST components can be combined to obtain a rotationally invariant set of texture features that can accurately classify a series of texture patterns. The DOST provides the computational efficiency and multi-scale information of wavelet transforms, while providing texture features in terms of Fourier frequencies. It outperforms leading wavelet-based texture analysis methods