There is a need for sensitive measures of disease progression in multiple sclerosis (MS) to monitor treatment effects and understand disease evolution. MRI measures of brain atrophy have been proposed for this purpose. This thesis investigates a number of measurement techniques to assess their relative ability to monitor disease progression in clinically isolated syndromes (CIS) and early relapsing remitting MS (RRMS). Presented, is work demonstrating that measurement techniques and MR acquisitions can be optimised to give small but significant improvements in measurement sensitivity and precision, which provided greater statistical power. Direct comparison of numerous techniques demonstrated significant differences between them. Atrophy measurements from SIENA and the BBSI (registration-based techniques) were significantly more precise than segmentation and subtraction of brain volumes, although larger percentage losses were observed in grey matter fraction. Ventricular enlargement (VE) gave similar statistical power and these techniques were robust and reliable; scan-rescan measurement error was <0.01% of brain volume for BBSI and SIENA and <0.04ml for VE. Annual atrophy rates (using SIENA) were -0.78% in RRMS and -0.52% in CIS patients who progressed to MS, which were significantly greater than the rate observed in controls (-0.07%). Sample size calculations for future trials of disease-modifying treatments in RRMS, using brain atrophy as an outcome measure, are described. For SIENA, the BBSI and VE respectively, an estimated 123, 157 and 140 patients per treatment arm respectively would be required to show a 30% slowing of atrophy rate over two years. In CIS subjects brain atrophy rate was a significant prognostic factor, independent of T2 MRI lesions at baseline, for development of MS by five year follow-up. It was also the most significant MR predictor of disability in RRMS subjects. Cognitive assessment of RRMS patients at five year follow-up is described, and brain atrophy rate was a significant predictor of overall cognitive performance, and more specifically, of performance in tests of memory. The work in this thesis has identified methods for sensitively measuring progressive brain atrophy in MS. It has shown that brain atrophy changes in early MS are related to early clinical evolution, providing complementary information to clinical assessment that could be utilised to monitor disease progression.

No description available.

Oligodendrocytes (OLs) are the cells responsible for myelin production in the central nervous system (CNS). Myelin serves to increase the efficiency of signal propagation down the axon and is essential for proper communication between the CNS and the periphery. As a result, pathologies affecting the OL, including multiple sclerosis (MS) and multiple system atrophy (MSA), present with a wide range of symptoms including impaired muscle control, loss of coordination, as well as cognitive deficits. While the biology of the OL continues to garner research interest, much remains to be understood about cell function in a healthy context, and also how the biology of these cells goes awry in disease. Our objective was to explore the effects of varying disease models on OL biology and use those findings to further our knowledge on the biology of OL development and regeneration. Here we explore OL function and dysfunction in the context of spinal muscular atrophy (SMA), MSA and MS. We have thoroughly characterized the OL response to SMN-depletion and have determined that SMN is not required for the development of OLs in the neonatal brain. Additionally, we have sought to characterize the endogenous role of MSA-disease relevant protein alpha-synuclein in OL development and have determined that this protein is not required for OL differentiation or CNS myelination. Lastly, we have explored the biology of the OL in the context of the inhibitory milieu it faces during remyelination in MS. We have investigated different pathways that may be involved in mediating signalling of one such inhibitory cue (chondroitin sulphate proteoglycans, CSPGs), and have extended this model to interrogate OL cytoskeletal dynamics in the context of CSPGs. Together, this work uses disease frameworks to investigate basic OL biology, as well as provides insights into how the OL and its interactions with the extracellular milieu should be considered in disease pathogenesis and therapeutic exploration.

The etiology of most neurodegenerative diseases remains a mystery. Environmental factors seemingly play an important role in neurodegenerative diseases, as does the immune system. Here, we describe immunological phenotypes associated with the neurodegenerative diseases AOA2 and ALS. During CSR, B-cells and neurons share a preferred pathway for DNA repair, NHEJ. SETX, the gene implicated in AOA2, has been implicated in the DDR. In SETX-deficient conditions, B-cells exhibit a defect in IgA class-switching as a result of impaired NHEJ and enhanced alternate-end joining, a slower process with higher mutational burden. These results suggest that neurons in SETX-deficient patients may also demonstrate defects in DNA repair, leaving them more susceptible to death. Further, IgA plays an important role in both protection from infections and as an anti-inflammatory mediator at mucosal surfaces. Defects in IgA class switching might lead to immune dysregulation in AOA2, similar to that observed in other neurodegenerative diseases. Viral infections have been associated with several neurodegenerative diseases, including ALS. However, a causal role for viruses in the etiology of ALS has never been established. Common viral infections can impact immune cell phenotypes within the CNS. Microglia are the primary immune cell of the CNS and are able to respond to subtle changes in the microenvironment. Microglia have neurotoxic properties upon hyper-activation. Influenza infection of SOD1G93A mice accelerated ALS disease progression and reduced overall survival. Exacerbated microgliosis was evident within the spinal cords of infected mice. Thus, the immune response stimulated by viral infections may result in toxic microgliosis that accelerates the progression of ALS. Together, this body of work describes a series of novel immunological abnormalities associated with AOA2 and ALS4. A deeper understanding of the role played by the immune system in patients with neurodegenerative disorders may unveil new targets for future therapies. / Thesis / Doctor of Philosophy (PhD) / The causes of most neurodegenerative diseases remain poorly understood. These diseases are highly complex, as they are influenced by both genetics and the environment and involve many different cell types. However, neurodegenerative diseases also share common hallmarks. The immune system is known to behave abnormally in several neurodegenerative diseases. Therefore, we set out to study the involvement of the immune system in two related neurodegenerative diseases, ataxia with oculomotor apraxia type 2 (AOA2) and amyotrophic lateral sclerosis (ALS). We demonstrate that AOA2-like mutations in senataxin (SETX) affect the formation of specific antibodies due to problems repairing broken DNA. We also find that viral infections activate immune cells within the spinal cord which can promote ALS progression. Taken together, this body of work suggests that the immune system plays an important role in AOA2 and ALS, and that drugs that prevent abnormal immune responses might help to treat these diseases.

Brain inflammation has been implicated with the pathogenesis of neurodegenerative diseases. Activated microglia and endothelial cells induce production of reactive oxygen species (ROS) and overexpress pro-inflammatory mediators that perpetuate tissue damage. Current treatments are not effective against progressive stages of neurodegenerative diseases and more advanced therapies need to be developed. Recently, nanomaterials have been investigated for therapeutic applications. Nanoparticles can increase efficiency of drug delivery due to increased tissue distribution and the ability to modify surface chemistry to increase biocompatibility and incorporate targeting moieties. In the present study, we established in vitro and in vivo brain inflammation models by administering lipopolysaccharide to mouse brain endothelial cells, microglia, macrophage cells and C57BL/6 male mice. Changes in mRNA expression of pro-inflammatory mediators were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), E-selectin, and intercellular adhesion molecule-1 (ICAM-1) displayed significant overexpression when compared to the control. Additionally, folate receptor-α (FR-α) was also overexpressed, confirming that our model will function appropriately for specific targeting experiments. Cellulose nanocrystals are rod-like particles, approximately 5 nm wide and 100-150 nm long. The surface area consists of extended hydroxyl groups and the structure is hydrophilic in nature. These characteristics make cellulose nanocrystals ideal for surface modification and ensuring long blood circulation half-life. Cell viability was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] conversion assay and a Lactate Dehydrogenase (LDH) Cytotoxicity Detection Kit. At each concentration of cellulose nanocrystals (10, 25, 50 μg/mL), both assays showed the nanoparticles to be non-toxic. Binding/uptake experiments utilizing a fluorescence plate reader and fluorescence microscope showed no non-specific uptake of untargeted cellulose nanocrystals. In contrast, when conjugated to folic acid, cellulose nanocrystals were selectively incorporated to folate receptor-overexpressing cells. These results indicate that both in vitro and in vivo brain inflammation models can be utilized to assess therapeutic efficacy of folate receptor-targeted bioconjugated nanoparticles. / Master of Science

"Oxidative stress is a type of cellular stress that can damage and kill cells. While it is naturally occurring, many non-natural substances found in our environment can also induce the formation of reactive oxygen species (ROS), which then cause oxidative stress within the cell. Oxidative stress has been shown to be involved in the death of neurons in a number of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis. The primary causes for these diseases are still unknown; however, we do know oxidative stress plays a primary role in their development. In conditions where oxidative stress is present, adenosine receptor expression has been upregulated and has played a cytoprotective role, but the specific mechanism of action is unknown. In this thesis, oxidative stress was studied in a model eukaryote, Saccharomyces cerevisiae, and the effects of the expression of the human A1 and A2A receptors upon stress response was examined. Oxidative stress was induced by the addition of hydrogen peroxide at concentrations of .5 mM, 1 mM, and 2 mM. The growth of cells expressing either A1-GFP R or A2A-GFP R at the varying hydrogen peroxide concentrations were compared to the parental cells. Confocal microscopy was performed to determine the receptor expression levels, and to confirm the expression of the receptors via their GFP tag. Immunoblots were also performed to assess the receptor expression level at the differing hydrogen peroxide concentrations. A ROS assay was also performed to show the presence of ROS and oxidative stress in the cells. No significant increase in receptor level expression or localization for either A1 R or A2A R at the varying hydrogen peroxide concentrations was found. The data did show trends indicating that A2A receptors may help process the oxidative stress better than A1 receptors and that A2A receptor containing cells had a shorter doubling time. However, more research on this subject should be performed in the future. However, the concentration of hydrogen peroxide should be greatly increased for further experiments in S. cerevisiae in order to better differentiate the trends observed." / 1 / Bryan Goldman

No description available.

Ubiquilin (UBQLN) is a member of type2 ubiquitin-like (UBL) protein family characterized by an UBL domain at the N-terminus and an ubiquitin associated (UBA) domain at the C-terminus. This protein has been shown to play an important role in the regulation of the levels, aggregation and degradation of various neurodegenerative disease-associated proteins. However, the specific functions and mechanisms of UBQLN regulation still remain to be elucidated. In this study, we investigate the effect of UBQLN expression on α-Amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor (AMPAR) degradation and the underlying molecular mechanisms. We show that UBQLN overexpression decreases AMPAR levels in neurons and also reduces GluA1 expression in HEK 293T cells. Moreover, our results indicate that UBQLN can form a complex with GluA1, and this interaction is related to the ubiquitination of AMPARs. In addition, we find a higher expression of UBQLN2 in Alzheimer’s disease (AD) patient brains, which might be a potential pathological mechanism of GluA1 reduction in AD. Given the crucial effect of UBQLN in AMPAR regulation, UBQLN may play an important role in synaptic transmission, brain functions as well as neurodegenerative diseases. / 2018-07-21T00:00:00Z

Neurodegenerative disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD), are increasingly common in our ageing society, are remain incurable. A major obstacle encountered by researchers in their attempts to find effective therapies is represented by the current lack of understanding of the molecular origins of these disorders. It is becoming clear that, although the aggregation of specific proteins, including amyloid β (Aβ) and tau in AD and α-synuclein in PD, hallmark these disorders, such behaviour is a consequence of a wider, system-level disruption of protein homeostasis. In order to identify the genetic factors contributing to such a disruption, the transcriptional changes that occur during neurodegenerative disease progression have received considerable scientific attention in recent years. In our approach, we considered another hallmark of these diseases - their characteristic patterns of spreading across the brain - to identify the nature of the transcriptional signature which underlies tissue vulnerability to protein aggregation. By understanding why tissues succumb in their characteristic sequential pattern in neurodegenerative diseases, and why some tissues remain almost completely resistant throughout, we hoped to obtain insight into the molecular origins of these disorders. Our results show that the AD progression can be predicted from a transcriptional signature in healthy brains related to the protein aggregation homeostasis of Aβ, tau, and the wider proteome. We highlight a relationship between a specific subproteome at high risk of aggregation (formed by supersaturated proteins), and the vulnerability to neurodegenerative diseases. We thus identify an AD-specific supersaturated set of proteins - termed the metastable subproteome, whose expression in normal brains recapitulates the staging of AD, with more vulnerable tissues having higher metastable subproteome expression. We find evidence of these vulnerability signatures transcending the tissue level of interrogation, with cellular and subcellular analysis also showing elevated levels of proteins known and predicted to predispose the aberrant aggregation of Aβ and tau. These results characterise the key protein homeostasis pathways in the inception and progression of AD, and establish an approach with the potential to be applied to other protein misfolding diseases, in the brain and beyond.

Matrix Metalloproteinase-28 (MMP-28) is the newest and least characterized member of MMP family. To date several potential substrate candidates for MMP-28 have been proposed but no in vivo substrates for this enzyme were confirmed. In the central nervous system (CNS) MMP-28 is believed to be important factor during myelination of the developing nervous system as well as during remyelination that follows neuronal injury. On the other hand, MMP-28 has been found in actively demyelinating lesions in both experimental autoimmune encephalopathy (EAE) and multiple sclerosis patients suggesting its possible role in pathological events associated with autoimmune neurodegenerative processes. In addition, MMP-28 has been linked to modulation of immune response and activation of macrophages which presents another role of this enzyme in autoimmune pathologies. In the study described herein, MMP-28 has been shown to affect myelin composition and appearance, mitochondrial protein content, and vesicular transport proteins. Moreover, the decrease in myelin basic protein quantity observed in healthy MMP-28KO animals affected the myelin staining intensity in various brain regions including corpus callous. Cellular energetic studies did not reveal differences in mitochondrial function in MMP-28KO animals and no difference in reactive oxygen species was observed. In the EAE model, MMP-28 deletion increased the occurrence of atypical form of EAE characterized by increased inflammation of arbor vitae of the brain. In addition, MMP-28 deletion decreased the inflammatory infiltrates present in brains obtained from EAE animals. Lastly, MMP-28 has been shown to affect cellular energetics and activation of bone marrow derived macrophages during the initial stages and after 24 h activation. In addition, MMP-28 deletion increased proinflammatory cytokines and receptors CD86 and iNOS found in M1 polarized macrophages. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection