Journal: Glia

Article Title: Semaphorin4A and H-ferritin utilize Tim-1 on human oligodendrocytes: A novel neuro-immune axis

doi: 10.1002/glia.23313

Figure Lengend Snippet: Characterization of isolated primary human oligodendrocytes. Isolated oligodendrocytes express CC1 (a) and MBP (b), both markers of mature myelinating oligodendrocytes. Furthermore, these cells also express Tim-1 (c). All images were taken at 63X magnification

Article Snippet: We used both primary human oligodendrocytes and the human cell line MO3.13, obtained from Cellutions Biosystems Inc. (Ontario, Canada) in the evaluation of Sema4A cytotoxicity and Hft uptake.

Techniques: Isolation

Journal: Glia

Article Title: Semaphorin4A and H-ferritin utilize Tim-1 on human oligodendrocytes: A novel neuro-immune axis

doi: 10.1002/glia.23313

Figure Lengend Snippet: Sema4A causes a dose dependent increase in apoptosis. (a and b) Sema4A causes cell death in mature, but not immature, human oligodendrocyte cell line MO3.13. (c) Sema4A causes cell death in primary human oligodendrocytes. (d) Annexin V positive, Propidium Iodide (PI) negative binding is increased in the presence of 40 μg/ml Sema4A (p = .0028, one-way ANOVA). (e) Caspases 3 and 7 are activated after Sema4A incubation in increasing doses. In all experiments, absorbance is quantified against the untreated or PBS controls. Means were evaluated for statistical significance using two-way ANOVA with Bonferroni’s post hoc comparisons. Values represent means of three biological replicates ± SD. *p < .05, **p < .01, ***p < .001

Article Snippet: We used both primary human oligodendrocytes and the human cell line MO3.13, obtained from Cellutions Biosystems Inc. (Ontario, Canada) in the evaluation of Sema4A cytotoxicity and Hft uptake.

Techniques: Binding Assay, Incubation

Journal: Glia

Article Title: Semaphorin4A and H-ferritin utilize Tim-1 on human oligodendrocytes: A novel neuro-immune axis

doi: 10.1002/glia.23313

Figure Lengend Snippet: Human oligodendrocytes take up H-ferritin. (a) A negative control quantum dot (QD) is not taken up by mature MO3.13 cells. (b) Mature MO3.13 cells take up H-ferritin labeled with a red fluorescent QD. (c) Primary human oligodendrocytes take up Hft-QD. CC1 staining represents mature oligodendrocytes while DAPI staining was used as a nuclear stain. All images were taken at 63X magnification

Article Snippet: We used both primary human oligodendrocytes and the human cell line MO3.13, obtained from Cellutions Biosystems Inc. (Ontario, Canada) in the evaluation of Sema4A cytotoxicity and Hft uptake.

Techniques: Negative Control, Labeling, Staining

Journal: Glia

Article Title: Semaphorin4A and H-ferritin utilize Tim-1 on human oligodendrocytes: A novel neuro-immune axis

doi: 10.1002/glia.23313

Figure Lengend Snippet: Human oligodendrocytes express Tim-1. (a) Luxol fast blue staining on human brain tissue demonstrating gray matter (GM) and white matter (WM) border, 2× magnification. (b) DAB-enhanced stain of Tim-1 in the same region, 10× magnification. (c) 40× magnification of Tim-1 stain, yellow arrows demonstrating classic “tram-track” oligodendrocyte morphology and arrangement. (d) Immunofluorescence colocalization between Tim-1 and CC1 demonstrating oligodendrocyte expression of Tim-1

Article Snippet: We used both primary human oligodendrocytes and the human cell line MO3.13, obtained from Cellutions Biosystems Inc. (Ontario, Canada) in the evaluation of Sema4A cytotoxicity and Hft uptake.

Techniques: Staining, Immunofluorescence, Expressing

Journal: Glia

Article Title: Semaphorin4A and H-ferritin utilize Tim-1 on human oligodendrocytes: A novel neuro-immune axis

doi: 10.1002/glia.23313

Figure Lengend Snippet: Sema4A from the CSF of patients with multiple sclerosis or HIV+ causes oligodendrocyte cell death. (a) CSF from patients with multiple sclerosis or HIV infection display a higher level of Sema4A. Scatterplot lines represent the mean values while box and whisker plot represents the median and interquartile range. (b) Sema4A isolated from the CSF of MS patients is cytotoxic to mature MO3.13 cells, comparable to the commercially available recombinant Sema4A (rSema4A). Negative control used in this study was PBS. (c) CSF before and after Sema4A immunoprecipitation was applied to mature MO3.13 cells, demonstrating that CSF with Sema4A causes cell death. Two-way ANOVA with Bonferroni’s post hoc comparison was used to assess statistical significance. Means are from three biological replicates ± SD, *p < .05, **p < .01, ***p < .001. (d) Complete CSF from HIV+ individuals applied at 100 μg/ml total protein to mature MO3.13 cells causes cytotoxicity, compared with CSF after Sema4A immunoprecipitation, recombinant Sema4A, and PBS control. Mann-Whitney test was used to assess statistical significance between the CSF with Sema4A and CSF minus Sema4A groups; *p = .0433. rSema4A and PBS controls are shown for comparison. (e) Individual differences in the mean cytotoxicity between each CSF sample. Differences were calculated as (Mean Absorbance of CSF with Sema4A)-(Mean Absorbance of CSF minus Sema4A)

Article Snippet: We used both primary human oligodendrocytes and the human cell line MO3.13, obtained from Cellutions Biosystems Inc. (Ontario, Canada) in the evaluation of Sema4A cytotoxicity and Hft uptake.

Techniques: Infection, Whisker Assay, Isolation, Recombinant, Negative Control, Immunoprecipitation, Comparison, Control, MANN-WHITNEY

Journal: Journal of Neuroinflammation

Article Title: Receptor tyrosine kinases play a significant role in human oligodendrocyte inflammation and cell death associated with the Lyme disease bacterium Borrelia burgdorferi

doi: 10.1186/s12974-017-0883-9

Figure Lengend Snippet: Differentiated human oligodendrocytes express RTKs. Immunofluorescence staining of MO3.13 cells for PDGFRβ ( a ), FGFR1 ( b ), and FGFR3 ( c ) is shown. Differentiated MO3.13 cells growing in chamber slides were exposed to either medium alone ( Med ) or B. burgdorferi ( Bb , MOI 10:1) for 48 h, fixed with paraformaldehyde as described in the “ ” section, and assessed for various receptors’ expression. Cells were additionally stained for MBP as a positive control, while the secondary antibody alone was used as a negative control (not shown). Bar represents 50 nm. A representative photograph from three to four experiments for each receptor is shown

Article Snippet: We also undertook to investigate the role of these receptors in primary human oligodendrocyte cells (ScienCell Laboratories Carlsbad, CA), which have also been characterized with respect to inflammation, apoptosis, and the role of the MEK/ERK pathway in Lyme neuroborreliosis [ , ].

Techniques: Immunofluorescence, Staining, Expressing, Positive Control, Negative Control

Journal: Journal of Neuroinflammation

Article Title: Receptor tyrosine kinases play a significant role in human oligodendrocyte inflammation and cell death associated with the Lyme disease bacterium Borrelia burgdorferi

doi: 10.1186/s12974-017-0883-9

Figure Lengend Snippet: Proposed summary of signaling mechanisms in MO3.13 human oligodendrocytes in response to B. burgdorferi . Activation of RTKs (EGFR/FGFR/PDGFR) by ligand-independent mechanisms, as mediated through GPCR or integrins, leads to phosphorylation of the intracellular domains of RTK. This leads to docking of proteins such as src, phospholipase Cγ (PLCγ) and others, which in turn activate downstream pathways like MAPK and MEK/ERK in particular. Significant activation of MEK/ERK, along with contribution from other MAPK and NFkB, leads to production of inflammatory mediators. We have demonstrated previously that MEK/ERK activation upregulates p53 and mitochondrial-mediated apoptosis in oligodendrocytes. The RTKs, for the sake of simplicity, are depicted as a single entity, although they are three separate receptors. Also, for the sake of illustration, they are surface located, although some could be trafficked internally . B. burgdorferi can bind integrins, which could transactivate RTKs. It also has well-known ligands to bind to TLR2 or TLR5. Although gram negative, it does not possess a typical LPS (which binds to TLR4) but rather a LOS. However, TLR2, TLR4, and TLR5 play minimal roles in induction of oligodendrocyte inflammatory mediators, as shown in this study. Thickness of the lines/arrows indicates signal strength. Dashed lines/arrows indicate unknown possibilities, while dashed and dotted lines/arrows indicate additional likely signaling mechanisms. Adapted from Parthasarathy and Philipp , by permission

Article Snippet: We also undertook to investigate the role of these receptors in primary human oligodendrocyte cells (ScienCell Laboratories Carlsbad, CA), which have also been characterized with respect to inflammation, apoptosis, and the role of the MEK/ERK pathway in Lyme neuroborreliosis [ , ].

Techniques: Activation Assay, Phospho-proteomics