Myelin is essential for normal nervous system conduction as well as providing metabolic support for the ensheathed axon and has been implicated to influence axon calibre (diameter of the axon body) growth. In demyelinating diseases, the disruption of these functions causes axon degeneration resulting in neurological impairment. The neurons that are myelinated in the CNS and the axon-oligodendrocyte (axon- OL) interactions that might regulate axon calibre and myelination during myelinated axon formation are still mostly unknown, preventing a deeper understanding of CNS development and repair. This doctoral thesis identifies a specific subset of interneurons that are myelinated and investigates the axon-oligodendrocyte interactions during axon calibre growth and initial myelination. In the zebrafish spinal cord, Commisural Primary Ascending interneurons (CoPA), Circumferential Descending interneurons (CiD) and reticulospinal neurons are amongst the first to be myelinated, whereas Commisural Bifurcating Longitudinal interneurons (CoBL) and Circumferential Ascending interneuron (CiA) are not myelinated during early developmental stages. Of the myelinated neurons, axon calibre of reticulo spinal neurons is increased in time with myelin ensheathment, while the axon calibre of CoPA and CiD interneurons is not increased with the onset of myelination. In order to investigate whether there might be a causative relationship between axon calibre increase and myelin ensheathment, the majority of oligodendrocytes were eliminated by olig2 morpholino knockdown. In the absence of oligodendrocytes, the axon calibre of reticulospinal neurons was normal, demonstrating that axon calibre growth is independent of axon-OL interactions and myelin ensheathment. In order to further investigate which aspects of myelinated axon formation might be regulated by axon-OL interactions, axonal activity was reduced through inhibition of synaptic vesicle release by global expression of Tetanus-toxin (TetTx). TetTx treated zebrafish showed a 40% decrease of myelinated axons in the spinal cord. Interestingly, only 10% of this reduction was caused by a decrease in oligodendrocyte number in the spinal cord. Single cell analysis of individual oligodendrocytes revealed a 30% reduction of myelin sheaths per oligodendrocyte in TetTx treated animals, indicating a positive correlation between synaptic vesicle release and the extent of myelination. Timelapse analysis of the myelinating behaviour of individual oligodendrocytes revealed that the decrease in myelin sheaths per cell in the absence of synaptic vesicle release results from a reduction in the initial formation of sheaths rather than an increased retraction of myelin sheaths. Furthermore, individual myelin sheaths formed by the same oligodendrocyte exhibit a dynamic range of different growth rates in control animals, which was reduced to a more uniform, slow growth of myelin sheaths in the absence of synaptic vesicle release. This suggests that local axon-OL interactions can regulate the dynamic myelin sheath growth through synaptic vesicle release. The analyses in this doctoral thesis identifies a subset of the neurons that are myelinated during the onset of myelination in the zebrafish spinal cord, demonstrates that axon caliber growth of these neurons is independent of myelin ensheathment and that axon-OL interactions mediated by synaptic vesicle release can regulate the extent of myelination and influence the dynamic myelinating behavior of oligodendrocytes in vivo. These findings begin to elucidate the axon-OL interactions underlying myelinated axon formation during CNS development, from which future studies might derive neuro-regenerative treatments for demyelinating diseases.

Oligodendrocytes are the myelinating cells of the central nervous system (CNS), whose function is to optimise neuronal transmission and preserving axonal integrity. Oligodendrocytes are derived from a stem cell population, called oligodendrocyte progenitor cells (OPCs). Oligodendrocyte lineage cells (OLCs) have been implicated in the pathophysiology of various diseases including not only demyelinating diseases (eg. Multiple Sclerosis (MS) or Pelizaeus-Merzbacher disease (PMD)), but also psychiatric disorders (eg. schizophrenia or Rett syndrome (RTT)). Regardless of the type of disease, understanding the underlying fundamental biology of the oligodendrocyte lineage cells is pivotal to develop therapeutic strategies. In the mouse embryonic forebrain OPCs are generated in consecutive waves from distinct brain regions along a spatiotemporal gradient; with ventral OPCs emerging before dorsal OPCs. The developmentally distinct OPCs, and their progenies, persist in the brain throughout life. To investigate whether ventrally and dorsally derived OLCs fulfil different functions in the adult brain, dorsally derived OPCs were ablated in development using a \textit{Sox10}-driven diphtheria toxin fragment A (DTA) mouse model. As dorsally derived OPCs populate the cortex, locomotor coordination and cognition were investigated following dorsal OPC ablation. Mice ablated of the dorsal OPC population do not show a significant deficit in learning and attentional function. In contrast, ablated mice show an impaired locomotor coordination, while general vigilance, gait, balance and sensation are comparable to control groups. The locomotor coordination disabilities are a result of alterations of brain, not spinal cord homeostasis, as only a neglect able number of OLCs in the spinal cord are affected by the ablation model. In addition, no signs of neuronal cell death or chronic inflammatory response was detected in response to the ablation. As the oligodendrocyte numbers are similar between control and ablated animals, the locomotor coordination phenotype can also not be explained by reduced number of oligodendrocytes. However, clustering analysis following single-cell Drop-sequencing uncovered a heterogeneity of oligodendrocyte (OL) subpopulations in the motor cortex. Whilst some OL subpopulations are of mixed developmental origin, others are exclusively formed by either ventrally or dorsally derived OLs, arguing that dorsal oligodendrocyte subpopulations are crucial for homeostatic brain function. In the absence of dorsal OPCs, ventral OPCs are not capable of forming dorsal oligodendrocyte subpopulations in response to dorsal OPC ablation. In conclusion, my results indicate a functional heterogeneity of developmentally-distinct oligodendrocytes in physiological brain function.

T2 relaxation imaging can be used to measure signal from water trapped between myelin bilayers; the ratio of myelin water signal to total water is termed the myelin water fraction (MWF). First, results from multi-component T2 relaxation and diffusion tensor imaging (DTI) were compared for 19 multiple sclerosis (MS) subjects at 1.5 T to better understand how each measure is affected by pathology. In particular, it was determined that the detection of a long-T2 signal within an MS lesion may be indicative of a different underlying pathology than is present in lesions without long-T2 signal. Next, the single-slice T2 relaxation measurement was implemented, refined, and validated at 3.0 T. Scan parameters were varied for phantoms and in-vivo brain, and changes in multi-exponential fit residuals and T2 distribution-derived parameters such as MWF were monitored to determine which scan parameters minimized artifacts. Measurements were compared between 1.5 T and 3.0 T for 10 healthy volunteers. MWF maps were qualitatively similar between field strengths. MWFs were significantly higher at 3.0 T than at 1.5 T, but with a strong correlation between measurements at the different field strengths. Due to long acquisition times, multi-component T2 relaxation has thus far been clinically infeasible. The next study aimed to validate a new 3D multi-component T2 relaxation imaging technique against the 2D single-slice technique most commonly used. Ten healthy volunteers were scanned with both the 2D single-slice and 3D techniques. MWF maps were qualitatively similar between scans. MWF values were highly correlated between the acquisition methods. Although MWF values were generally lower using the 3D technique, they were only significantly so for peripheral brain structures, likely due to increased sensitivity of slab-selective refocusing pulses used for the 3D approach. The 3D T2 relaxation sequence was then applied to the study of MS to take advantage of the increased brain coverage. Thirteen MS subjects and 11 controls underwent T2 relaxation and DTI examinations to produce histograms of MWF and several DTI-derived metrics. MS MWF histograms differed considerably from those of controls, and differences in MS MWF histograms did not mirror differences in DTI histograms relative to matched controls. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

Multi-echo T2 relaxation measurements of the human spinal cord (SC) reveal a short T2 pool of water believed to arise from water trapped between myelin bilayers, where the proportion of this water to the total water signal is called the myelin water fraction (MWF). In the present study, MWF were measured in the healthy human cervical spine at the C4-C6 vertebral levels in vivo using a 3D modified 32 echo CPMG sequence to acquire axial slices perpendicular to the cord. Volunteers were recruited in two age ranges, under 30 years old and over 50 years old, and a subset of both groups were scanned twice to test reproducibility. Mean MWF in the dorsal and lateral column WM of the group under 30 years of age was 0.29 (0.01) (mean(SE)), which agrees with previously reported MWF values in the cervical spine. The mean absolute difference between two scans was 0.06 or 26%. A negative correlation between WM MWF and age was hinted at in these findings, however more subjects are required to improve statistical power. This study paves the way for the use of 3D myelin water imaging in the cervical spine at 3.0T for the assessment of SC WM pathology. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

The interplay between oligodendrocyte (OL) and extracellular matrix (ECM) is critical to the proper maturation of this unique cell type. Recent work has established the β1 integrin-signaling pathway, a mediator for ECM/OL interactions, as an essential component of myelin sheath formation in the central nervous system (CNS). A major downstream effector of β1 integrin is integrin-linked kinase (ILK), an adaptor and structural platform protein. Herein, we (1) generated a model system to study ILK in vivo and (2) employed the model to elucidate ILK’s role in regulating OL biology. To assess the importance of ILK in OL-mediated myelination, we ablated ILK in primary OLs. ILK loss delayed morphological maturation and led to filamentous actin accumulation in the processes and cell body. Further, we noted an upregulation in RhoA activity, with pathway inhibition rescuing an OL subset. We next moved our studies in vivo. First, we assessed the proteolipid protein promoter’s utility as OL-specific Cre driver. Protocols established, we generated an ILK conditional knockout line (Ilk cKO). Ultrastructural analysis of Ilk cKO optic nerves revealed increased number of amyelinated nerve fibers at P14 with subsequent recovery by P28. The observed transient defects were due neither to a loss nor a gain in total number of mature or progenitor OLs. To rationalize recovery, we grew ILK-depleted OLs on an “inert” substrate. Here, while morphology improved, ILK-depleted OLs were characterized by enlarged and sluggish growth cones as well as microtubule disorganization. Taken together, our data suggests a role for ILK in regulating the morphological maturation of OLs both in vitro and in vivo, the loss of which results in defective OL branching and membrane formation with phenotype and subsequent recovery dependent upon niche complexity.

Multiple sclerosis (MS) is a debilitating disease in which demyelinated lesions form in the central nervous system (CNS). A specific microRNA, miR-145-5p, is dysregulated both in blood samples from RRMS patients and in chronic lesions from progressive MS patients. In the context of remyelination, miR-145-5p regulation may be important as it exhibits strong differential regulation in oligodendrocytes (OLs), the myelinating cells of the CNS, and is also expressed in other CNS glial cell types. Dysregulation of miR-145-5p may therefore play into pathologies observed in both relapsing-remitting (RRMS) and progressive MS. Using pre-clinical rodent models, we aimed to determine how altering normal expression of miR-145-5p specifically affects OL maturation, and how the dysregulation observed in MS may affect various aspects of disease. First using a miR-145 knockdown model in primary rat OLs, we found in vitro that miR-145-5p plays a role both in maintaining oligodendrocyte progenitor cells (OPCs) in their proliferative state and preventing premature differentiation to OLs and that knockdown of miR-145 in OLs enhanced their differentiation. These effects were due at least in part to miR-145-5p regulation of a critical myelin gene transcription factor. The effects of miR-145-5p were further assessed in a miR-145 knockout mouse model in vivo. Contrary to in vitro assays, enhanced myelination was not detectable during development in these animals, nor when remyelination was assessed using the cuprizone toxic model of acute demyelination. However, chronic cuprizone exposure resulted in striking remyelination and functional recovery in miR-145 deficient animals. Sparse remyelination in wild-type animals with chronic cuprizone exposure was concomitant with upregulation of miR-145-5p, which was not the case with acute exposure, identifying miR-145-5p dysregulation as a unique feature of chronic demyelination. Specific assessment of miR-145-5p overexpression in OLs in vitro resulted in severe differentiation deficits and eventual apoptosis, driven molecularly by altered expression of multiple pathways critical to successful OL differentiation and subsequent myelination. Finally, we induced an inflammatory model of demyelination, experimental autoimmune encephalomyelitis (EAE), in our miR-145 knockout mouse to assess the role of miR-145-5p in autoimmune-mediated myelin damage. The clinical severity of EAE in miR-145 deficient animals was reduced, and this was accompanied by reduced loss of myelin and lessened immune cell infiltration in miR-145 knockout spinal cords. Alterations in both astrocytic and microglial activation were detected with loss of miR-145, suggesting that improved clinical outcomes in this model may be underpinned by changes in EAE-mediated neuroinflammation. Collectively, these data suggest that miR-145-5p plays differing roles in both progressive and inflammatory MS, affecting multiple glial cell types in the CNS. Excitingly, loss of miR-145 expression in our mouse model of chronic demyelination allowed extensive remyelination and functional recovery following chronic demyelination, and in EAE improved clinical outcomes driven by underlying improvements in myelin retention and altered neuroinflammatory reactions. Thus, miR-145-5p merits further investigation as a potential therapeutic target to help overcome both remyelination failure in all forms of progressive MS and inflammation-driven demyelination in RRMS and early secondary progressive MS (SPMS).

Multiple sclerosis is a demyelinating disease affecting the central nervous system. It is the most prevalent disabling neurological condition among young adults, with onset typically between 20 and 40 years of age. Infiltrating immune cells and microglia activations are associated with inflammatory and neurodegenerative mechanisms. Current available disease modifying therapies suppress or modulate the immune system. These pharmaceuticals differ with respect to administration route and frequency, adverse effects, and efficacy. This paper provides a thorough manuscript illustrating the major prescribing factors, efficacy profiles, adverse events, and contraindications that patients and clinicians should consider while choosing a treatment. Despite the advancements made over the past 20 years, patients with progressive multiple sclerosis have few therapeutic options. Additionally, this paper assesses emerging therapies and disease targets on the pharmaceutical horizon, which have shown promise for all disease phenotypes.

Introduction: Bipolar disorder is associated with cortical abnormalities, including deficits in intracortical myelination. Intracortical myelin follows an inverted-U trajectory over the lifetime, but this trajectory is blunted in individuals with bipolar disorder. Little is understood about which genetic factors contribute to these deficits. Neuregulin-1, a cell-signalling protein, has been shown to contribute to cortical abnormalities and increase susceptibility to related disorders. Assessing the prevalence of neuregulin-1 polymorphisms, notably rs6994992, in bipolar disorder may elucidate the genetic contributors of intracortical myelin deficits and increase our understanding of factors causing susceptibility to bipolar disorder. Methods: 67 participants with bipolar disorder type I and 75 healthy control participants were included. T1-weighted MRI images were collected and processed to create R1 cortical maps, a proxy measure of intracortical myelin. Participant blood samples were genotyped at the rs6994992 locus. Linear models were used to test whether intracortical myelin can be predicted by age, bipolar diagnosis and NRG1 genotype. Results: Intracortical myelin is significantly predicted by age, diagnosis and genotype together in the motor cortex (left: R2 = 0.09, p < 0.01, right: R2 = 0.06, p < 0.05), the right premotor cortex (R2 = 0.095, p < 0.001), and the right inferior frontal cortex (R2 = 0.098, p < 0.001). Age is a significant individual predictor of intracortical myelin in the right dorsal anterior cingulate cortex, the bilateral motor cortex, the right premotor cortex, and the right inferior frontal cortex. Conclusions and Future Directions: Our study suggests that the right premotor, bilateral primary motor, and right inferior frontal cortices are regions of interest for understanding how intracortical myelin changes throughout the lifetime, especially in bipolar disorder. Future work should examine the impact of polygenic risk scores of bipolar disorder on intracortical myelin. / Thesis / Master of Science (MSc) / Bipolar disorder is associated with neurobiological changes, including cortical abnormalities, contributing to a greater disorder burden. Cortical myelination changes throughout the lifetime and larger deficits are found in individuals with bipolar disorder. However, the role of genetics in these intracortical myelin deficits is largely unknown. This thesis investigates how intracortical myelin content in various regions of the cortex is impacted by age, bipolar disorder diagnosis, and neuregulin gene variants. The goal of this research is to contribute to a better understanding of how genetics and age impact intracortical myelin in bipolar disorder to better understand the neurobiological changes of the disorder.

Myelin is a protein complex which plays an integral role in developing and maintaining proper brain function. Due to the plasticity of the brain, and the dynamic nature of myelin, it is critical to develop methods that allow for the investigation of changes in myelin in vivo, to further our understanding of the brain. A substantial amount of myelin is found in the grey matter (GM) of the cerebral cortex – the outermost structure of the brain that supports higher order functions including cognition and more fundamental functions, such as sensation and motor control. While in vivo investigations have traditionally used imaging to focus on myelin in the deep white matter (WM) tracts in the brain, advances in magnetic resonance imaging (MRI) are now allowing investigations of intracortical myelin (ICM). The research in this thesis presents methodology for investigating intracortical myelin levels using magnetic resonance imaging (MRI) in humans, with the aim of developing a better understanding of how myelin contributes to healthy cortical function, and how it may be disrupted in disease. To characterize intracortical myelin, a novel MRI analysis technique was developed early in this work to report the thickness of the heavily myelinated and lightly myelinated layers of the cortex. This measure of myelinated cortical thickness uses a clustering algorithm to separate the layers of the cortex based on voxel intensity in a T1- weighted (T1W) MRI with strong intracortical contrast. The resulting myelinated thickness maps match known myelin profiles of the brain, with cortical regions such as the primary visual and motor cortices displaying proportionally thicker, heavily myelinated layers. The utility of the myelinated cortical thickness for answering clinical questions was tested in bipolar disorder, where a preferential loss of the more myelinated layers in the dorsal lateral prefrontal cortex was found. This study provided the first in vivo evidence of ICM disruptions in bipolar disorder. Later in the thesis work, after surface-based analysis techniques became available, an alternative approach to investigate intracortical myelin was developed that sampled the T1W image intensity at a calculated depth of the cortex as a measure of myelin content. This methodology was used for studying the association of ICM with age in healthy adults ranging from late adolescence to middle-adulthood. It was found that three cortical depths followed a similar trajectory through this age-span, reaching their peak between 35 and 40 years of age. This study contributes to a picture of ICM amounts increasing well into middle age in healthy adults and provides a baseline for studies investigating how this may be disrupted in disease Up to this point, the analysis in the thesis used a specialized T1W MRI that had been optimized to provide strong intracortical contrast, but a question remained of how useful the technique would be if more commonly collected clinical MRIs were used as inputs. This analysis was thus applied to standard T1W and T2-weighted (T2W) anatomical MRIs to test its clinical applicability. 360 participants were investigated from the TRACK-HD dataset to test if intracortical signal analysis could follow the progression of Huntington’s disease. A significant increase in intracortical T1W/T2W signal was found in the most advanced disease group in several cortical regions. This increase in intracortical signal is likely tracking a known increase in iron and/or myelin levels in the Huntington’s disease brain. However, this work suggests that ICM studies would best be conducted with optimized imaging to better be able to characterize the subtle ICM variations within the GM. Overall, the work in this thesis presents two techniques for whole-brain mapping of the distribution of intracortical myelin using MRI. The clinical applicability of the techniques was demonstrated in examples of mental and neurodegenerative disorders. The future directions of this work include developing imaging specific to either myelin or iron as well as revisiting these problems while imaging at greater resolution to better characterize the laminar profile across the cortex. / Thesis / Doctor of Philosophy (PhD)

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