Jacqueline Quandt

Gangliosides are glycolipids abundantly expressed on the extracellular layer of several cells in the central nervous system. Gangliosides influence cell-cell interactions, neuronal development, as well as axonal growth and stability and are targets of autoimmunity in several neurological disorders. Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease attributed to the depletion of lipids and myelin components. Anti-ganglioside antibodies (AGA) have been studied in MS, yet their relevance to disease processes remains poorly characterized.Our objective was to compare serum AGA levels between healthy controls (HC) and participants with either a single demyelinating event known as clinically isolated syndrome (CIS), or confirmed MS of relapsing-remitting (RRMS), secondary progressive (SPMS), or primary progressive (PPMS) MS types. Enzyme-linked immunosorbent assays (ELISA) were used to measure levels of antibodies to the gangliosides GA1, GM1, GM2, GD1a, GD1b, and GQ1b. Levels of each AGA measured were not significantly different between HCs and CIS or any MS subgroup. However, they were generally negatively associated with age and there were correlations between several AGAs in MS participants. Predictive modeling demonstrated an inverse relationship between AGAs to GM1 and GD1b, where the likelihood of having MS is increased with higher levels of anti-GM1 and lower levels of anti-GD1b. Higher levels of anti-GD1b and lower levels of anti-GM1 were associated with a greater likelihood of being in the CIS group. Our study showed that patterns and interactions exist between AGA levels amongst different MS groups and that analyzing a single ganglioside is of limited value. The predictive potential of AGA levels to GM1 and GD1b in distinguishing MS type highlight the potential of AGA signatures to aid the discerning disease course and shed light on the underlying pathophysiology of MS.

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Background: The process of axonal degeneration and neuronal loss has been described as the major cause of irreversible clinical disability in multiple sclerosis (MS). An ideal neuroprotective strategy would be to focus on inhibition of axonal degeneration and on protection against neuronal cell death in addition to immunomodulation. The aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) is a protein with neuroprotective properties previously described in ischemic insults and oxidative damage. We hypothesize that alterations in ARNT2 expression are associated with changes in cell viability in in vitro and in vivo models of multiple sclerosis. Methods: Following exposure to various compounds mimicking MS disease processes, ARNT2 protein and mRNA levels were observed in primary cortical neuron-enriched cultures using western blotting, quantitative polymerase chain reaction (qPCR) and immunocytochemistry, alongside cytotoxicity measurements, using a lactate dehydrogenase (LDH) release assay/Live/Dead® Viability/Cytotoxicity assay. ARNT2 protein levels were also evaluated in primary cortical astrocytes using immunocytochemistry. Analyses in an animal model of MS, experimental autoimmune encephalomyelitis (EAE) were conducted, with tissue collected at various stages of the disease course, to examine ARNT2 expression patterns in vivo. Results: Examination of individual neurons reveals that most cells demonstrate low-medium ARNT2 expression under steady-state conditions. Exposing cells to both low and higher concentrations of hydrogen peroxide (H₂O₂) to mimic mild to more severe oxidative stress significantly increases ARNT2 protein levels early, as measured via western blotting and immunocytochemistry. At the mRNA level, oxidative stress fails to drive Arnt2. This increased detection of ARNT2 protein is observed in both neurons and reactive astrocytes specifically within the neuronal-enriched mixed populations. Non-reactive astrocytes also express ARNT2 at baseline conditions. Finally, ARNT2 is differentially expressed in healthy versus EAE tissue at peak disease. Conclusions: This work demonstrates for the first time that ARNT2 can follow altered expression patterns in vitro in neurons depending on the severity/duration of the stimulus involved in MS disease progression. This lays a foundation for understanding the link between ARNT2 expression and neuronal health in vitro.

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Microglia are the primary immune cells found within the central nervous system (CNS), playing a vital role in neuronal function, trophic support and also modulating immune or inflammatory responses to pathogens or damage during disease. Microglia are essential to repair processes influencing axonal health and remyelination. However, the study of microglia is limited as significant yields of microglia through tissue culture are difficult to obtain. We show that the addition of granulocyte macrophage colony-stimulating factor (GM-CSF) during the culture of embryonic microglia yields significantly greater cell numbers. GM-CSF cultured microglia exhibit a non-differentiated phenotype similar to in vivo microglia and represent a useful model for disease and reparative processes in the CNS. Using our primary microglial model, we investigated two proteins, Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) and receptor-mediated endocytosis – 8 (RME-8). ARNT2, a transcription factor for several proteins but most notably for the neuronal growth factor, brain derived neurotrophic factor (BDNF), has been primarily studied in neurons. Our studies show regulation of ARNT2 in astrocytes and immune cells (microglia and splenocytes) under inflammatory conditions. In the experimental autoimmune encephalomyelitis (EAE) model, splenocytes exhibited lower ARNT2 expression than those from healthy controls. Lipopolysaccharide and interferon-γ increased otherwise low ARNT2 expression in microglia.RME-8 is a protein that is important in endosomal trafficking. Mutations in RME-8 have been linked to Parkinson’s disease and essential tremor. However, RME-8 has yet to be characterized within the CNS. Motor neurons, astrocytes and ependymal cells expressed RME-8 in healthy control mice; RME-8 was increased and co-localized with CD68 positive cells in immune infiltrates in EAE mice. Our results show the uptake of dextran in RME-8 mutant knock-in microglia is decreased, indicating the importance of this protein in phagocytic processes. These results show that microglia can be effectively cultured from embryonic tissue with the addition of GM-CSF in comparison to previously established protocols and are similar to microglia in vivo. Furthermore, inflammatory mediators influence expression of ARNT2 and RME-8 and may highlight roles for each in neuroprotection or phagocytic function respectively, thereby influencing inflammatory neurodegenerative or reparative processes relevant to several diseases in the CNS.

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Background: Reactive oxygen and nitrogen species are implicated in inflammatory-mediated damage to the central nervous system in multiple sclerosis (MS) and an animal model of the disease, experimental autoimmune encephalomyelitis (EAE). We have shown that oral administration of the antioxidant TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), a stable nitroxide radical, lowers incidence and reduces severity of disease in EAE. We hypothesize that TEMPOL limits inflammatory demyelinating disease by regulating the development of pathogenic immune responses that influence immune cell activation, including T cell and antigen presenting cell phenotypes and function. Methods: Immune responses were compared between control and TEMPOL-fed EAE or healthy mice by examining differences in proliferation, population distribution, surface marker expression, and cytokine production in immune cells isolated from lymphoid organs. The effect of added TEMPOL on immune cell proliferation and phenotype was also studied in vitro using mixed lymphocyte reactions (MLR) with human or mouse cells, and in isolated murine lymphoid cell cultures stimulated with anti-CD3. Results: TEMPOL-fed animals exhibit comparable levels of myelin-reactive T cells versus controls, but show reduced production of the pro-inflammatory cytokines interferon gamma, tumor necrosis factor alpha, and transforming growth factor-beta 1. Flow cytometry showed enrichment of CD8+ over CD4+ T cells in lymphoid tissues of TEMPOL-fed EAE mice, as well as decreased MHC II and increased CD80 and CD86 expression in myeloid cells and myeloid dendritic cell (DC) populations. Enrichment of Foxp3+ regulatory T cells was also observed in lymph nodes with TEMPOL. In vitro, iii TEMPOL was found to enhance proliferation of lymphoid cells in mouse MLR or when stimulated with anti-CD3 in a dose-dependent manner. Human MLR experiments also showed enhanced cell proliferation and enrichment of CD8 T cells in the presence of TEMPOL. Adding TEMPOL to cell cultures decreased expression of MHC II, CD80, and CD86 in splenic myeloid cells and myeloid DCs. Conclusions: These studies suggest that TEMPOL is not globally immunosuppressive, but instead alters the phenotype of antigen-specific or autoreactive immune cells generated in vivo, reducing the pro-inflammatory nature of immune responses in EAE. These immunomodulatory properties contribute to TEMPOL’s potential as an efficacious therapeutic in MS.

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SPARC (secreted protein acidic and rich in cysteine) is a cell-matrix modulating protein involved in angiogenesis and endothelial barrier function, yet a potential role in cerebrovascular repair and inflammatory responses in the central nervous system (CNS) has not previously been characterized. The inflammatory demyelinating disease, multiple sclerosis (MS) is characterized pathologically by inflammatory infiltrates, demyelination and axonal damage/loss and aberrant alterations in blood-brain barrier (BBB) integrity. We hypothesize that SPARC expression may be influenced by inflammatory or repair processes during MS, and that SPARC itself may influence BBB integrity. This study examined SPARC expression in cultured human cerebral microvascular endothelial cell (hCMEC/D3), an in vitro model of the BBB, under steady state conditions or those modeling an inflammatory milieu by immunoblotting and immunocytochemistry. hCMEC/D3s constitutively express SPARC during proliferative growth and downregulate SPARC as cells establish a BBB phenotype. SPARC expression in cerebral endothelia directly correlated with the cell proliferation marker Ki-67, consistent with a role for SPARC in CNS angiogenesis. Proinflammatory cytokines associated with inflammation and immune activation differentially regulate SPARC expression in cerebral endothelia. Tumor necrosis factor alpha (TNF-α) cytokine or lipopolysaccharide (LPS) endotoxin treatment significantly increased SPARC protein levels. TNF-α and interferon gamma (IFN-γ) cotreatment abrogated SPARC induction compared to TNF-α alone, suggesting divergent roles for each cytokine in regulating SPARC expression in cerebral endothelia. Compared to cultures replenished with media lacking exogenously supplied SPARC, addition of a physiological SPARC concentration observed in healthy individuals (0.1μg/ml) increased tight junction protein expression of zonula occludens 1 (ZO-1) and occludin by approximately thirty percent, suggesting a role in BBB maintenance. Paradoxically, functional assays show recombinant human SPARC applied exogenously increased the transendothelial permeability of hCMEC/D3 monolayers. In agreement, barrier hCMEC/D3s exposed to increased SPARC concentrations (1-10 μg/ml) associated with pathological conditions in vivo, reduced ZO-1 and occludin by one-third. Together, these data support a role for SPARC in BBB maintenance under normal physiological conditions and BBB alterations during inflammatory conditions. In this regard, SPARC levels may play a key role in regulating BBB integrity and serve to alter processes of CNS inflammation and repair.

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