Browsing by Author "Ousman, Shalina S."

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    Aging exacerbates myelin disruption and axon injury following demyelination
    (2019-04-30) Michaels, Nathan J.; Yong, Voon Wee; Ousman, Shalina S.; Casha, Steven
    For the majority of individuals with multiple sclerosis, the disease begins with a relapsing-remitting disease course that transitions to secondary progressive multiple sclerosis. Population-based studies have identified age as a critical risk factor for this transition. The mechanisms linking age and progressive multiple sclerosis remain poorly understood. Studies in animal models of demyelination suggest age is associated with increased axon injury and loss. Despite these findings, how several features and mechanisms of lesion development in the spinal cord white matter differ between young and aging animals are unclear. In this thesis, I characterized the early events of lesion formation in young and middle-aged animals, focusing particularly on axon injury/loss, oligodendrocyte lineage cell loss, and myelin disruption. I used an unbiased, RNA sequencing approach to explore the mechanisms underlying age-exacerbated injury. I used differential gene expression and pathway analysis data acquired from RNA sequencing to hypothesize that reactive oxygen species derived from NADPH oxidase is associated with elevated myelin and axon damage in middle-aged animals. I then used a transgenic mouse approach to differentiate between microglia and infiltrating peripheral myeloid cells in the spinal cord and found gp91phox, the catalytic subunit of NADPH oxidase, was primarily expressed in microglia and its expression was elevated in middle-aged microglia. Lastly, I tested the ability of indapamide, a generic medication with antioxidant properties, to ameliorate age-exacerbated myelin and axon loss in vivo, and to scavenge reactive oxygen species derived from microglia in vitro. Indapamide attenuated the degree of myelin disruption and axon loss, decreased lipid peroxidation in the spinal cord white matter, and reduced extracellular superoxide produced by microglia.
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    Chondroitin sulfate proteoglycans as novel mediators of inflammation in multiple sclerosis
    (2018-07-23) Stephenson, Erin Laurel; Yong, Voon Wee; Ling, Chang-Chun; Ousman, Shalina S.; Proud, David
    The extracellular matrix of the central nervous system is an interconnected network of proteins and sugars. The extracellular matrix is crucial for homeostasis, but its remodeling in diseases impacts both injury and repair. Here I introduce an extracellular matrix member that participates in immune-matrix interactions, the chondroitin sulfate proteoglycans. Chondroitin sulfate proteoglycans integrate signals from the microenvironment to activate immune cells, interact with their receptors, and bind signaling molecules such as cytokines and chemokines. Chondroitin sulfate proteoglycans are commonly upregulated in a variety of neuroimmune and peripheral inflammatory diseases, and are key drivers of pathogenesis. In this thesis, I characterized the upregulation of chondroitin sulfate proteoglycans in the inflammatory demyelinating mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis. One novel observation was the upregulation of chondroitin sulfate glycosaminoglycans and the chondroitin sulfate proteoglycan member versican V1 at peak inflammation, as well as their accumulation in the perivascular cuff, a portal of entry of immune cells into the central nervous system. Chondroitin sulfate proteoglycans augmented the inflammatory activity of macrophages, enhancing the production of pro-inflammatory cytokines, matrix-degrading enzymes, and promoting their migration and phagocytosis. I explored the various mechanisms underlying this pro-inflammatory response. I found that receptors involved in the response to chondroitin sulfate proteoglycans may include toll-like receptor -2 and -4, and downstream signaling molecules include the adaptors MyD88 and TRIF as well as NFkB and protein kinase C. Lastly, I screened novel fluorinated glucosamine and xyloside compounds for their ability to interfere with chondroitin sulfate proteoglycan production and in other assays pertinent to multiple sclerosis. I found the compound, Ac-4,4-diF-GlcNAc, was extremely potent at reducing CSPG synthesis by astrocytes, promoting oligodendrocyte precursor cell growth on an inhibitory matrix, and had non-toxic but anti-inflammatory capabilities. Following experimental autoimmune encephalomyelitis, Ac-4,4-diF-GlcNAc treatment significantly reduced clinical disease score in multiple treatment paradigms, and reduced the number of infiltrating leukocytes in the spinal cord. This thesis highlights novel roles of chondroitin sulfate proteoglycans in inflammation, details mechanisms for their effects on immune cells, and also highlights a novel compound that may be beneficial for targeting chondroitin sulfate proteoglycans to control inflammatory cascades.
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    Combination of Hydroxychloroquine and Indapamide to Attenuate Neurodegeneration in Models of Multiple Sclerosis
    (2020-06-11) Brown, Dennis; Yong, Voon Wee; Ousman, Shalina S.; Koch, Marcus A.
    As the underlying pathophysiology of progressive forms of multiple sclerosis (MS) are unclear, current treatment strategies remain inadequate. Progressive MS is associated with increased oxidative stress and neuronal damage in lesions along with an extensive representation of activated microglia/macrophages. We tested a novel combination of generic medications, hydroxychloroquine (HCQ) and indapamide, to target the aforementioned pathologies associated with progressive MS. HCQ is an anti-malarial known to inhibit microglial activation, ameliorate EAE disease activity, and is currently in a Phase II trial in primary progressive MS. Indapamide is an antihypertensive previously discovered in our laboratory drug screen to be an antioxidant. Indapamide was also found to be neuroprotective in vitro and in the lysolecithin model of demyelination. As MS disease activity is ongoing or episodic, I employed a pre-treatment paradigm prior to inducing a demyelinating spinal cord lesion in mice with lysolecithin. I found the combination but not individual treatment of HCQ and indapamide to reduce microglia/macrophage CD68+ representation in lesion, and to attenuate neuronal impairment as determined by amyloid precursor protein accumulation in axons that traverse the area of injury. In vitro, HCQ and indapamide in combination had robust neuroprotective and antioxidant effects, in addition to attenuating microglial activation. With a lack of pharmaceutical treatments currently available for the progressive forms of MS, it is critical that research is focused on improving therapeutics for this unmet need of MS. With the promising results of the combination treatment of HCQ and indapamide in neuroprotection in vitro and in vivo, this thesis hopes to provide the preclinical rationale for a new and affordable combination treatment strategy targeting multiple facets of progressive MS.
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    Cystatin C and sex dimorphism in experimental autoimmune encephalomyelitis
    (2018-08-16) Hoghooghi, Vahid; Ousman, Shalina S.; Pittman, Q. J.; Jirik, Frank Robert
    This dissertation characterizes the role of a lysosomal enzyme inhibitor, Cystatin C (CysC), in an animal model of multiple sclerosis (MS), called experimental allergic encephalomyelitis (EAE). MS is an autoimmune disease characterized by infiltration of myelin reactive immune cells into the central nervous system (CNS) that is more prevalent in genetically susceptible women compared to men. Alteration in the level of CysC in the CNS of MS patients is known, but its role has not been determined. Towards clarifying the role of CysC in EAE, I initially hypothesized that CysC would have a beneficial function in EAE due to its protective role in another autoimmune disease, collagen-induced arthritis. Unexpectedly, using C57BL/6 and CysC null (CysCKO) mice, I discovered that CysC plays a detrimental role in female but not male EAE mice. This sex dimorphism in the function of CysC could also be observed in immune cell infiltration and demyelination in EAE. Towards elucidating the cellular mechanism(s) underlying the sex difference driven by CysC in EAE, I discovered that the protease inhibitor promotes the activation of antigen presenting cells such as macrophages in females that subsequently mediates stimulation of CD4+ T cells. The T cell activation-promoting ability of CysC in female macrophages appears to be related to antigen presentation in macrophages since molecular mediators of antigen processing and loading (LC3II) and presentation (CD80, CD86, MHC II) were disrupted in female, but not male, macrophages that lacked CysC. Cumulatively, these observations indicate that CysC drives sex dimorphism in macrophages. Lastly, to understand why CysC would induce sex differences in macrophage function and thus EAE susceptibility, I investigated if sex hormones were involved. I found that CysC plays a detrimental role in EAE in the presence of estrogen. This dissertation thus provides novel evidence that CysC has a role in mediating sex dimorphism in EAE.
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    Designing a novel 3-D in-Vitro scaffold to define mechanisms underlying neuronal myelination
    (2018-11-28) Shahidi, Sahar; Syed, Naweed I.; Sanati Nezhad, Amir; Ousman, Shalina S.; Yusuf, Kamran
    All nervous system functions in animals require neuronal assembly during development and the ensuing communications between large networks of neurons, which are often difficult to monitor in the intact brain. As such, most labs around the globe have opted to use and explore in vitro model systems where neurons are generally grown on two-dimensional, plane glass substrates, which limit the ability to decipher fundamental understanding of the mechanisms underlying neuronal growth, polarity and synapse specificity. Most approaches used today employ 2-D models where neurons are cultured on the Poly-D-lysine (PDL) coated substrate which does not mimic the 3-D configuration of the intact mammalian brain – thus limiting a direct comparison between in vivo and in vitro conditions. Assessing cellular and molecular mechanisms of neuronal myelination are critical to determine how myelination and demyelination processes occur in vertebrate models so as to understand developmental and neurodegenerative diseases such as multiple sclerosis. However, there are no suitable in vitro models available to date whereby the process of axon myelination could be studied directly at the level of individual central and peripheral neurons. In contrast to PDL, collagen offers a 3-D structure in which neurons can be suspended in a 3-D configuration, allowing glia to gain access to axonal membrane to exhibit myelination. However, we still lack a reliable 3-D model where mechanisms of neuronal polarity and myelination could be studied at the level of individual peripheral and central neurons. In this study, I designed a 3D substrate comprising of a gelatin base hydrogel with tunable chemical mechanical properties. Using rat Dorsal Root Ganglia Cells (DRG) and their corresponding Schwann cells (SC), I compared and contrasted the effectiveness of GelMA with PDL and Collagen substrates and provide the first direct evidence that the former is more conducive to studying myelination than the later two. Moreover, I also demonstrate that both DRG growth and SC behavior on GelMA resembles to what is seen in vivo thus validating further the usefulness of this substrate for future studies.
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    Elucidating the role of cystatin c in astrocytes in multiple sclerosis
    (2020-08-20) Palmer, Alexandra L.; Ousman, Shalina S.; Yong, Voon Wee; Antel, Jack P.; Kurrasch, Deborah M.; Kuipers, Hedwich F.; Jirik, Frank Robert
    Multiple sclerosis (MS) is one of the most common neurological disabilities of young adults. During the disease course there are a variety of molecules that become dysregulated, one of them being cystatin C (CysC). CysC is a small molecule inhibitor of cysteine cathepsins and is implicated in a variety of biological processes. The role of CysC in MS remains undetermined. Astrocyte reactivity is well noted in the MS literature and contributes to pathological processes during disease development and progression. This thesis set out to 1) characterize the expression of CysC in MS and the animal model, experimental autoimmune encephalomyelitis (EAE), 2) clarify the role of CysC in astrocytes and, 3) determine whether CysC can be therapeutically targeted. CysC expression was characterized in both MS and EAE central nervous system (CNS) samples. The expression of CysC was found to be increased in both MS and EAE brain and spinal cord samples, its expression overlapped with astrocytes, and a loss of CysC (CysCKO) in EAE ameliorated disease. This attenuation of clinical disability in CysCKO EAE mice was associated with a CNS intrinsic effect. A mixed glial culture system was then used to explore the effect of CysC on the response of astrocytes to pro-inflammatory stimuli. From this, it was found that CysCKO glia reduced cytokine and chemokine production in response to lipopolysaccharide (LPS) and increased cytokine and chemokine production upon activation by Interleukin-1beta + interferon-gamma, however CysC may not mediate this effect through the astrocyte signalling pathways STAT1, STAT3, or NF-kappaB. Lastly, Valsartan, a generic medication with the ability to reduce serum CysC levels in humans, was examined to see if it could therapeutically decrease CysC levels in vivo during EAE. Valsartan decreased CysC levels and delayed disease onset, however its effect did not appear to be mediated through astrocytes. Finally, an astrocyte-specific CysC conditional KO mouse was developed to more directly interrogate the role of CysC in astrocytes in vivo during EAE. This thesis provides novel evidence that CysC may influence astrocyte cytokine and chemokine secretion and may be a potential therapeutic target in MS.
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    The glycosyltransferase EXTL2 and its regulation of remyelination and neuroinflammation
    (2019-04-11) Pu, Yu; Yong, Voon Wee; Biernaskie, Jeff A.; Ousman, Shalina S.
    The extracellular matrix is an area that is poorly studied in the context of central nervous system (CNS) regeneration and inflammation. Previous works have demonstrated the chondroitin sulfate proteoglycans (CSPGs) to be a major group of matrix constituents that potently inhibit the growth of axons both during development and following injury. However, there are gross changes that occur in the composition of the matrix in injury, the full implications of which have yet to be elucidated. We use the lysolecithin-induced model of demyelination in mice to investigate the roles that CSPGs serve in remyelination and inflammation with respect to microglia and macrophages. We find that CSPGs are a mediator of microglia/macrophagemediated inflammation in the spinal cord, and loss of a regulatory enzyme, exostosin-like 2 (EXTL2), results in exacerbated neuroinflammation following injury. In culture, bone marrow-derived macrophages from EXTL2-/- animals produce more matrix metalloproteinase and tumor necrosis factor alpha when stimulated with CSPGs. The supernatant from these cells are also more neurotoxic to cultured neurons. Overall, this work highlights CSPGs as an important factor that influences inflammation in the CNS.
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    Investigating the Heat Shock Protein 110 as a Modifier Of Prion Infection In Vitro and In Vivo
    (2021-11-22) Marrero Winkens, Cristóbal; Schätzl, Hermann M; Trang, Tuan; Braun, Janice E. A.; Ousman, Shalina S.; Nussbaum-Krammer, Carmen
    Prion diseases are fatal transmissible neurodegenerative disorders affecting humans and wild or domesticated animals. Molecularly, they are caused by a misfolding of the cellular prion protein (PrPC) into a highly pathogenic isoform (PrPSc). Over time, PrPSc aggregates into fibrils which are fragmented to recruit and convert further PrPC. Even though this process is essential to the replication of PrPSc, the molecular players involved remain unknown. Based on reports showing that the molecular chaperone Hsp110 forms part of a mammalian disaggregation machinery, we hypothesize that Hsp110 is critical for the fragmentation of PrPSc in prion propagation. To test this, Hsp110 levels were manipulated in prion-infected cultured cells: Transient knock-down was found to reduce PrPSc levels in neuronal and non-neuronal cells, while transient overexpression increased PrPSc in a dose-dependent manner in neuronal cells. Knockout of Hsp110 by CRISPR/Cas9 reduced the susceptibility of neuronal cells to prion infection. Overall, the effects of Hsp110 manipulation in cultured cells are consistent with an involvement of Hsp110 in prion propagation. Next, mice overexpressing Hsp110 (TgApg-1 mice) were inoculated with the 22L and Me7 prion strains. Compared to WT mice, TgApg-1 mice showed significantly prolonged survival after Me7- but not 22L-inoculation. To test whether this prolongation of survival occurred due to altered PrPSc propagation, the biochemical features of PrPSc were examined in terminally-diseased animals. No difference was found between PrPSc of WT or TgApg-1 mice, suggesting that Hsp110 overexpression may have been protective independently of PrPSc. Finally, the effect of compounds broadly inducing the expression of heat shock proteins was examined. WT FVB mice inoculated with RML or Me7 prions were treated with either celastrol, geranylgeranylacetone or vehicle. No difference in survival time was found between the groups, despite evidence of heat-shock response induction in drug-treated animals. These results suggest that broad heat-shock induction is not protective in prion infection. Overall, our in vitro studies are consistent with a role of Hsp110 in PrPSc fragmentation, but our in vivo work is inconclusive in this regard. We, therefore, propose further research aimed at exploring prion fragmentation and the role of Hsp110 in prion infection.
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    Limiting Autoimmune Neuroinflammation using Novel T Cell Suppressants & Investigating Overlapping Lysosomal Reductase Function in Macrophages and Osteoclasts
    (2019-07-19) Ewanchuk, Benjamin Walter; Yates, Robin M.; Ramachandran, Rithwik; Ousman, Shalina S.; Riabowol, Karl T.
    Aberrant activation of adaptive immune cells can culminate in autoimmune diseases such as multiple sclerosis (MS). Recently, potent anti-inflammatory properties for the cooling compound, icilin, and its receptor target, Transient Receptor Potential Melastatin-8 (TRPM8), were characterized in the context of inflammatory colitis—the first half of this dissertation describes an attempt to repurpose the anti-inflammatory qualities of icilin and TRPM8 for the treatment of lymphocyte-mediated neuroinflammation. We found icilin treatment strongly attenuated experimental autoimmune encephalomyelitis (EAE), a murine model of MS, via an unexpected TRPM8-independent mechanism. Icilin inhibited the proliferation, polarization and downstream effector function of CD4+ T cells, suggesting a promising drug for limiting neuroinflammation. Citing the advantageous pharmacodynamics of icilin, we additionally screened a library of icilin-related analogues for anti-proliferative character. We identified several lead compounds with improved anti-proliferative properties compared to icilin in vitro and preliminary efficacy in vivo limiting EAE severity. Collectively, this work characterizes a new class of T cell suppressants while emphasizing clear off-target effects for the cooling drug icilin beyond TRP channel activation. Antigen presenting cells such as macrophages process phagocytosed cargo within the highly degradative phagolysosome, facilitating antigenic stimulation of T cells and adaptive immunity. The redox microenvironment of the phagolysosomal lumen regulates several phagolysosomal biochemistries, including proteolysis and disulfide reduction. The lysosomal enzyme γ-interferon-inducible lysosomal thiol reductase (GILT) directly catalyzes disulfide reduction and enhances proteolysis by thiol-dependent cysteine cathepsins within the phagolysosome—while clearly implicated in antigen processing, secondary roles for GILT remain largely undefined. The second half of this dissertation establishes a role for GILT in osteoclasts, bone resorbing cells derived from macrophages. GILT expression was highly upregulated in osteoclasts in response to osteoclastogenic and inflammatory cytokines, and GILT-deficient mice were discovered to be osteopetrotic. In vitro, GILT-deficient osteoclasts demonstrated a reduced capacity to resorb bone. GILT not only directly reduced disulfides within bone matrix structural proteins, but also enhanced the activity of cathepsin K, the prototypical osteoclast collagenase. Thus, this work reveals a novel, non-immunological role for GILT in osteoclast function and bone turnover.
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    Minocycline treatment timing and its influence on serotonin expression following spinal cord injury
    (2019-11) Flood, Jennifer Margaret; Whelan, Patrick J.; Trang, Tuan; Casha, Steven; Ousman, Shalina S.
    The effects of incomplete traumatic spinal cord injury (SCI) can be partly reversed by the plasticity of local and spared descending projections. A promising window of plasticity occurs for a number of weeks following injury and involves the control of neuroinflammatory processes. The FDA-approved drug, minocycline, is a promising drug for treating SCI since it decreases microglia activity, reduces macrophage activity, and generally provides neuroprotective properties. In this thesis I established a timeline of injury, looking at both serotonin (5-HT) and microglia/macrophage (Iba-1) immunoreactivity (ir), and I targeted a time point before a significant reduction of descending serotonergic fibers, in the form of 5-HTir, took place (i.e. 1-week). I found that the administration of minocycline increased 5-HTir caudal and ipsilateral to the lesion, compared to shams and controls. Using the selected time point, 1-week post-SCI, I administered minocycline and found a decrease in lesion size and an increase of 5-HTir both caudal and ipsilateral to the injury as well as rostral and contralateral to the injury. In this thesis, I provide evidence that minocycline impacts 5-HT expression when administered acutely and one week following SCI. These data suggest that the timing of minocycline treatment influences the neuroprotective properties previously reported and also influences descending 5-HT expression post-SCI.
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    Plxna4 and its role in neuronal survival in the developing zebrafish hindbrain
    (2019-09-17) Nurcombe, Zachary W.; McFarlane, Sarah; Kurrasch-Orbaugh, Deborah M.; Ousman, Shalina S.; Mains, Paul E.
    Plexins are a family of transmembrane proteins that bind Semaphorin ligands, and are known to function in axon guidance. In embryonic zebrafish, I find that plxna4 is expressed widely in the nervous system early in development, and localizes to the hindbrain as neurogenesis and differentiation proceed. In the hindbrain of a plxna4-/- CRISPR mutant, expression of the Islet transcription factor by cranial motor neurons appears depleted at 48 hours post fertilization, with increased apoptosis at 24 hpf. These data support a survival role for Plexina4 in the embryonic zebrafish hindbrain. Literature indicates that the secreted heat shock protein Clusterin is a candidate ligand to mediate apoptosis through Plexina4, as Clusterin is known to promote cell survival, and bind Plxna4. I find that clu is expressed by the floorplate of the embryonic zebrafish hindbrain. Epistasis experiments reveal Clusterin and Plexina4 promote cell survival in the developing zebrafish hindbrain via independent pathways.
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    Understanding Brain Injury-Induced Immunosuppression and the Relationship to the Development of Infection
    (2018-06-05) Scott, Brittney Noelle Vivian; Kubes, Paul; Zygun, David A.; Kramer, Andreas H.; Ousman, Shalina S.; Schryvers, Anthony B.; Fox-Robichaud, Alison E.
    Infection is a leading cause of morbidity and mortality among hospitalized patients. It has become increasingly apparent that patients with neurological injury have an increased risk for infection due to secondary immunodeficiency. Previous work from our research group found a novel role for invariant natural killer (iNKT) cells in stroke-induced immune suppression, characterized by a shift from a Th1- to Th2-dominant systemic cytokine profile and an increased risk for infection. This work better defined the crosstalk that occurs between the brain and systemic immune system after ischemic stroke, however, many questions remained and whether similar mechanisms were involved in other types of brain injury was unclear. Thus, we evaluated the relationship between iNKT cells, Th1 and Th2 systemic cytokine profiles, and the development of infection among critically ill patients with traumatic brain injury and haemorrhagic stroke. We found that these patients had significantly subnormal levels of many immune mediators, including IFN-γ and TNF-α, indicative of systemic immune suppression. Moreover, iNKT cells were activated among these patients and positively associated with plasma Th2/Th1 cytokine ratios. Infection was common and occurred among forty-six percent of the patients. Additionally, we used animal models to investigate traumatic brain injury-induced immune modulation and its relationship to infection. We observed rapid activation of iNKT cells in the circulation and a >2-fold increase in plasma Th2/Th1 cytokine ratios, which peaked at 8 hours after injury. Remarkably, we also observed rapid changes in the lung microenvironment induced by traumatic brain injury, which influenced the outcome after infection. Moreover, in an attempt to better understand the epidemiology of infection among patients with traumatic brain injury, we conducted a systematic review of the world’s literature on this topic. We summarize and discuss the reported occurrence rates of infection, and the microbiology and risk factors associated with different types of infection, among patients hospitalized after traumatic brain injury. This thesis provides new insights into the relationship between brain injury and the development of infection. Understanding the unique risk for infection after acute brain injury will ultimately translate to better prevention and treatment regimens for these patients.