Journal: Biomolecules

Article Title: Model Senescent Microglia Induce Disease Related Changes in α-Synuclein Expression and Activity

doi: 10.3390/biom8030067

Figure Lengend Snippet: Expression of the potassium channel Kv1.3. Protein extracts were prepared from control and iron-fed C8B4 microglia. Western blot analysis was carried out to determine the level of Kv1.3 in the microglia. Bands for the protein were observed in both control and iron-fed microglia. Levels of tubulin were also determined to verify protein loading. The results showed a significant ( p < 0.05) elevation of Kv1.3 in iron-fed microglia. Shown are the mean and S.E.M. of four separate experiments.

Article Snippet: Anti- l -ferritin mouse monoclonal (SC-25616, Santa Cruz, Dallas, TX, USA) was used at 1:5000, anti-Kv1.3 rabbit polyclonal was used at 1:400 (APC101, Alomone, Jerusalem, Israel), and anti- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mouse monoclonal was used at 1:2000 (6C5, Abcam).

Techniques: Expressing, Western Blot

Journal: Journal of Neuroinflammation

Article Title: A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation

doi: 10.1186/s12974-017-0906-6

Figure Lengend Snippet: LPS-activated BV2 microglia upregulate Kv1.3 channels. a Immunofluorescence microscopic comparison of Kv1.3 protein expression (detected by Anti-Kv1.3 rabbit pAb 1:500) by unstimulated and LPS-activated (100 ng/mL × 24 h) BV2 microglia. b Western blot confirmation of LPS dose-dependent increase in Kv1.3 protein in BV2 whole-cell lysates (Anti-Kv1.3 pAb 1:1000). c Immunofluorescence microscopic comparison of cell surface Kv1.3 channels in unstimulated and LPS-activated BV2 cells using ShK-F6CA. d Flow cytometric comparison of ShK-F6CA-labeled cell surface Kv1.3 channels and microglial activation marker ICAM-1 in unstimulated and LPS-activated BV2 cells. e Assessment of microglial activation markers MHCII and CD69 in unstimulated and LPS-activated BV2 cells

Article Snippet: The immunoblots were incubated with anti-Kv1.3 rabbit polyclonal antibody (APC 101, Alomone labs, 1:1000) and anti-beta-actin mAb (Cell Signaling, #3873,1:1000) for 24 h and subsequently appropriate fluorescent secondary antibodies (anti-mouse IgG IRDye® 800 conjugate, Rockland, 1:20,000 and anti-rabbit IgG Alexa Fluor® 680 conjugate, Invitrogen) were added for 1 h. An Odyssey Scanner (LI-COR) was used to visualize labeled proteins.

Techniques: Immunofluorescence, Expressing, Western Blot, Labeling, Activation Assay, Marker

Journal: Journal of Neuroinflammation

Article Title: A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation

doi: 10.1186/s12974-017-0906-6

Figure Lengend Snippet: In vivo demonstration of Kv1.3 channel upregulation by microglia and brain-infiltrating macrophages in a mouse model of LPS-induced neuroinflammation. a LPS was administered to adult C57BL/6 mice by tail vein injection, and brain mononuclear cells were analyzed by flow cytometry (CD11b, CD45, ShK-F6CA, and ICAM-1). b Most cells were CD11b + CD45 low microglia while small populations of brain-infiltrating macrophages CD11b + CD45 high and CD11b - non-myeloid cells were observed. c – e Comparison of Kv1.3 channel and ICAM-1 expression in brain mononuclear cells isolated from control and LPS-treated mice in microglia ( c ), brain-infiltrating macrophages ( d ), and CD11b - subpopulations ( e ) (* p < 0.05, ** p < 0.01, *** p < 0.005)

Article Snippet: The immunoblots were incubated with anti-Kv1.3 rabbit polyclonal antibody (APC 101, Alomone labs, 1:1000) and anti-beta-actin mAb (Cell Signaling, #3873,1:1000) for 24 h and subsequently appropriate fluorescent secondary antibodies (anti-mouse IgG IRDye® 800 conjugate, Rockland, 1:20,000 and anti-rabbit IgG Alexa Fluor® 680 conjugate, Invitrogen) were added for 1 h. An Odyssey Scanner (LI-COR) was used to visualize labeled proteins.

Techniques: In Vivo, Injection, Flow Cytometry, Expressing, Isolation

Journal: Journal of Neuroinflammation

Article Title: A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation

doi: 10.1186/s12974-017-0906-6

Figure Lengend Snippet: Identification of novel Kv1.3 channel-dependent molecular mechanisms by microglial quantitative proteomics. BV2 microglia were exposed to PBS, LPS (100 ng/mL), ShK-223 (100 nM), or LPS+ShK-223 for 24 h (3 replicates/group) and whole-cell lysates were used for mass-spectrometric analysis. a Volcano-plot: Of 450 proteins differentially expressed across all 4 groups ( black dots ), 144 proteins were significantly up- or downregulated following LPS exposure ( top-right and top-left quadrants; vertical dotted lines represent the upper and lower boundaries of 1.25-fold change threshold while the horizontal dotted line represents p value threshold of 0.05 comparing LPS vs. control groups). Among these, ShK-223 reversed the LPS effect in 21 proteins (highlighted in red ). b LPS-upregulated proteins that were reversed by ShK-223 and c LPS-downregulated proteins reversed by ShK-223 are shown. Relative expression represents label-free quantitation data in each treatment condition normalized to the control PBS group. The dotted horizontal line represents the twofold change threshold as compared to the PBS group. d – f Circle plots representing results from GO analyses are shown ( gray : protein/gene symbol, red : biological process, green : cellular component, purple : molecular function). Only significantly enriched GO terms (unadjusted p ≤ 0.05 based on enrichment score) are shown (* p < 0.05, ** p < 0.01, *** p < 0.005). d GO analysis of 21 LPS-regulated Kv1.3-dependent proteins ( n = 21, stringent p ≤ 0.05 threshold comparing LPS vs. LPS+ShK-223). e GO analysis of LPS-upregulated Kv1.3-dependent proteins ( n = 26, p ≤ 0.10 threshold comparing LPS vs. LPS+ShK-223). f GO analysis of LPS-downregulated Kv1.3-dependent proteins ( n = 40, p ≤ 0.10 threshold comparing LPS vs. LPS+ShK-223)

Article Snippet: The immunoblots were incubated with anti-Kv1.3 rabbit polyclonal antibody (APC 101, Alomone labs, 1:1000) and anti-beta-actin mAb (Cell Signaling, #3873,1:1000) for 24 h and subsequently appropriate fluorescent secondary antibodies (anti-mouse IgG IRDye® 800 conjugate, Rockland, 1:20,000 and anti-rabbit IgG Alexa Fluor® 680 conjugate, Invitrogen) were added for 1 h. An Odyssey Scanner (LI-COR) was used to visualize labeled proteins.

Techniques: Expressing, Quantitation Assay

Journal: Journal of Neuroinflammation

Article Title: A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation

doi: 10.1186/s12974-017-0906-6

Figure Lengend Snippet: Kv1.3 channels regulate microglial taxis and formation of F-actin complexes induced by LPS. a In the gap closure assay of microglial taxis, BV2 cells were grown to near confluence followed by placement of a uniform scratch using a 200-μm pipette tip. Ability of microglia to close this gap was assessed by measuring the percentage of gap closure over a 24-h period. Paired (0 and 24 h) representative images from each treatment group are shown ( a , left ), and the comparison of percentage gap closure (over 24 h) is shown ( a , right ) (six replicates per condition). b BV2 microglial transmigration across a transwell membrane (8-μm pore diameter) after exposure to control, ShK-223, ShK-186, LPS or LPS+ShK-223, or ShK-186, towards serum-containing medium (10% fetal bovine serum). Following 24 h of transmigration, cells were detached from undersurface of the insert (0.25% Trypsin), and cells that successfully migrated across the membrane were counted on a hemocytometer ( n = 3, independent experiments). c Comparison of F-actin containing focal adhesion complexes in BV2 microglia following exposure to control, ShK-223, LPS, or LPS+ShK-223. Fixed and permeabilized BV2 cells were labeled with phalloidin-rhodamine to detect F-actin ( left : immunofluorescence images). The number of focal complexes were counted per cell at ×40 magnification ( right ) and compared (>25 cells counted per condition). d DCFDA assay of ROS production by brain mononuclear cells isolated from C57BL/6 mice treated with PBS, LPS, ShK-223, or LPS+ShK-223 IP for four consecutive days ( n = 3, mice/group). Cells were loaded with DCFDA for 30 min and assayed for ROS activity by flow cytometry. e Flow cytometric phagocytosis assay of fluorescent (PE) microbeads by brain mononuclear cells isolated from C57B6/L mice treated with PBS, LPS, ShK-223, or LPS+ShK-223, n = 3/group). Dotted line : fluorescence of cells not exposed to beads; gray histogram : PBS-treated; black histogram : LPS-treated. The proportions of all phagocytic cells and highly phagocytic cells were compared across treatment groups (* p < 0.05, ** p < 0.01, *** p < 0.005)

Article Snippet: The immunoblots were incubated with anti-Kv1.3 rabbit polyclonal antibody (APC 101, Alomone labs, 1:1000) and anti-beta-actin mAb (Cell Signaling, #3873,1:1000) for 24 h and subsequently appropriate fluorescent secondary antibodies (anti-mouse IgG IRDye® 800 conjugate, Rockland, 1:20,000 and anti-rabbit IgG Alexa Fluor® 680 conjugate, Invitrogen) were added for 1 h. An Odyssey Scanner (LI-COR) was used to visualize labeled proteins.

Techniques: Transferring, Transmigration Assay, Labeling, Immunofluorescence, Isolation, Activity Assay, Flow Cytometry, Phagocytosis Assay, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation

doi: 10.1186/s12974-017-0906-6

Figure Lengend Snippet: Kv1.3 channels regulate TAP1, EHD1, GABPA, and IL1B expression in primary murine microglia. a Experimental plan for in vivo studies: adult C57BL/6 mice received four daily IP doses of PBS, ShK-223, LPS, or LPS+ShK-223, and brain mononuclear cells were isolated for flow cytometry from one hemisphere and qRT-PCR studies from the other hemisphere ( n = 3, mice/group). b Results from flow cytometric studies measuring intracellular EHD1 protein expression in freshly isolated CNS MPs (gated first on live cells, followed by CD11b + CD45 low and CD11b + CD45 high populations). At least 10,000 live CNS MPs were counted per sample. Left: Example of flow cytometric histograms comparing EHD1 expression in CD11b + CD45 low microglia from one mouse from each treatment group. Right: Quantitative analysis of EHD1 + cells in CD11b + CD45 low and CD11b + CD45 high populations. c Results from pRT-PCR studies measuring mRNA expression of TAP1, EHD1, GABPA, and IL1B in CNS MPs. For these studies, all CNS MPs isolated from one hemisphere were used for RNA extraction in Trizol, followed by cDNA preparation followed by quantitative PCR. HPRT was used as the housekeeping gene. Data normalized to HPRT were then normalized to PBS-treated control samples (* p < 0.05, ** p < 0.01, *** p < 0.005).

Article Snippet: The immunoblots were incubated with anti-Kv1.3 rabbit polyclonal antibody (APC 101, Alomone labs, 1:1000) and anti-beta-actin mAb (Cell Signaling, #3873,1:1000) for 24 h and subsequently appropriate fluorescent secondary antibodies (anti-mouse IgG IRDye® 800 conjugate, Rockland, 1:20,000 and anti-rabbit IgG Alexa Fluor® 680 conjugate, Invitrogen) were added for 1 h. An Odyssey Scanner (LI-COR) was used to visualize labeled proteins.

Techniques: Expressing, In Vivo, Isolation, Flow Cytometry, Quantitative RT-PCR, RNA Extraction, Real-time Polymerase Chain Reaction

Journal: Journal of Neuroinflammation

Article Title: A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation

doi: 10.1186/s12974-017-0906-6

Figure Lengend Snippet: LPS-induced MHCI trafficking in microglia involves Kv1.3 channels. a Comparison of MHCI and MHCI-related protein expression in BV2 proteomic data. Normalized LFQ data (compared to control-treatment) is shown (three replicates/group). b Experimental plan for in vivo studies: Adult C57BL/6 mice received four daily IP doses of PBS, ShK-223, LPS, or LPS+ShK-223 and brain mononuclear cells were isolated for flow cytometry (pooled n = 9/group). c Comparison of MHCI (H2Kb) expression (median fluorescence intensity) in CD11b + CD45 low microglia. d Representative flow cytometry frequency histograms of MHCI expression in CD11b + CD45 low microglia. e Comparison of MHCI expression in CD11b + CD45 high brain-infiltrating macrophages. f Comparison of the proportions of CD11b + CD45 high CNS macrophages (among all CD11b + cells) in the four treatment groups (* p < 0.05, ** p < 0.01, *** p < 0.005)

Article Snippet: The immunoblots were incubated with anti-Kv1.3 rabbit polyclonal antibody (APC 101, Alomone labs, 1:1000) and anti-beta-actin mAb (Cell Signaling, #3873,1:1000) for 24 h and subsequently appropriate fluorescent secondary antibodies (anti-mouse IgG IRDye® 800 conjugate, Rockland, 1:20,000 and anti-rabbit IgG Alexa Fluor® 680 conjugate, Invitrogen) were added for 1 h. An Odyssey Scanner (LI-COR) was used to visualize labeled proteins.

Techniques: Expressing, In Vivo, Isolation, Flow Cytometry, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation

doi: 10.1186/s12974-017-0906-6

Figure Lengend Snippet: Kv1.3 blockade inhibits LPS-induced MHCI-restricted antigen presentation to CD8 T cells. a Experimental plan for MHCI-restricted T cell proliferation studies: BV2 microglia were treated in vitro with PBS, ShK-223 (100 nM), LPS (100 ng/mL), or LPS+ShK-223 for 24 h (four replicates/group) and then loaded with 2 μg/mL Ova (257-264) peptide for 30 min at 37 °C. Washed microglia were co-cultured with CFSE-labeled splenic T cells from Ova (257-264)-specific OT-1 mice for 48 h (25,000 microglia:150,000 T cells/well). For in vivo experiments, adult wild-type mice were treated with four daily IP doses of saline, ShK-223, LPS, or LPS+ShK-223 ( n = 6/group) and isolated brain mononuclear cells were used for in vitro T cell proliferation studies. After 48 h, CFSE dilution (leftward shift in CFSE staining) was assessed by flow cytometry as a measure of CD8 + T cell proliferation. b Comparison of proliferating CD8 + cells across various treatment groups from in vitro and in vivo studies. Right : Representative frequency histograms from in vivo experiments. No direct effects of LPS or Ova peptide on T cells were noted without the presence of microglia. c Flow cytometric detection of CD3 + CD11b − T cells in brain mononuclear cells after thorough cardiac perfusion. Mice were injected (IP, once daily for 4 days) with PBS, LPS, ShK-223, or LPS+ShK-223. Panel a shows the gating strategy. After forward and side-scatter gating for live mononuclear cell population, CD11b − CD3 + cells were gated and assessed for T cell markers (CD3, CD8) and activation/cytolytic marker CD95 (Fas). d Flow cytometric evidence for increased CD8 + T cell trafficking to the brain following LPS treatment. e Comparison of proportions of brain CD8 + and CD8 − T cell populations across the 4 treatment groups. f Comparison of CD95 (Fas) expression in brain CD8 + and CD8 − T cell populations across treatment groups (* p < 0.05, ** p < 0.01, *** p < 0.005)

Article Snippet: The immunoblots were incubated with anti-Kv1.3 rabbit polyclonal antibody (APC 101, Alomone labs, 1:1000) and anti-beta-actin mAb (Cell Signaling, #3873,1:1000) for 24 h and subsequently appropriate fluorescent secondary antibodies (anti-mouse IgG IRDye® 800 conjugate, Rockland, 1:20,000 and anti-rabbit IgG Alexa Fluor® 680 conjugate, Invitrogen) were added for 1 h. An Odyssey Scanner (LI-COR) was used to visualize labeled proteins.

Techniques: In Vitro, Cell Culture, Labeling, In Vivo, Isolation, Staining, Flow Cytometry, Injection, Activation Assay, Marker, Expressing

Journal: Journal of Neuroinflammation

Article Title: A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation

doi: 10.1186/s12974-017-0906-6

Figure Lengend Snippet: Identification of Kv1.3-regulated signaling mechanisms in LPS-induced microglial activation. a , b Canonical pathway analysis of 120 Kv1.3-dependent proteins identified in our proteomic dataset revealed highly represented signaling pathways, two of which are shown here (see Additional file : Table S5 for others). Members of this list of 120 proteins are marked with ( red circle ). Transcription factors are also highlighted ( transparent red circle ). Arrows indicate directionality of the interaction (upstream vs. downstream). a GABPA, a Kv1.3-dependent transcription factor that was downregulated by LPS, was placed upstream of several Kv1.3-regulated proteins. b MHCI proteins of relevance to our results (TAP1, Tapasin, and EHD1 proteins) were represented in a signaling network that suggested that STAT1 and IRF1 may serve as upstream regulators of these proteins. c Comparison of normalized protein expression of transcription factors identified in our proteomic dataset across treatment groups. d Quantitative RT-PCR data comparing IRF1, IRF7, and NFKB1 mRNA expression across treatment groups (paired t tests were used for these comparisons; six replicates/group). e Phospho-flow cytometric studies of serine (S727) and tyrosine (Y701) STAT1 phosphorylation in BV2 microglia ( n = 5/treatment group) at 30-min and 3-h time points. f Immunofluorescence microscopy showing partial co-localization between Kv1.3 ( green , detected by ShK-F6CA labeling) and CD14 ( red ) in LPS-activated BV2 microglia (* p < 0.05, ** p < 0.01, *** p < 0.005)

Article Snippet: The immunoblots were incubated with anti-Kv1.3 rabbit polyclonal antibody (APC 101, Alomone labs, 1:1000) and anti-beta-actin mAb (Cell Signaling, #3873,1:1000) for 24 h and subsequently appropriate fluorescent secondary antibodies (anti-mouse IgG IRDye® 800 conjugate, Rockland, 1:20,000 and anti-rabbit IgG Alexa Fluor® 680 conjugate, Invitrogen) were added for 1 h. An Odyssey Scanner (LI-COR) was used to visualize labeled proteins.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Microscopy, Labeling

Journal: Journal of Neuroinflammation

Article Title: A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation

doi: 10.1186/s12974-017-0906-6

Figure Lengend Snippet: Cartoon showing the proposed mechanism by which Kv1.3 channel function regulates LPS-induced EHD1 and TAP1 upregulation and MHC I trafficking an antigen presentation by microglia

Article Snippet: The immunoblots were incubated with anti-Kv1.3 rabbit polyclonal antibody (APC 101, Alomone labs, 1:1000) and anti-beta-actin mAb (Cell Signaling, #3873,1:1000) for 24 h and subsequently appropriate fluorescent secondary antibodies (anti-mouse IgG IRDye® 800 conjugate, Rockland, 1:20,000 and anti-rabbit IgG Alexa Fluor® 680 conjugate, Invitrogen) were added for 1 h. An Odyssey Scanner (LI-COR) was used to visualize labeled proteins.

Techniques: