Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: (A) IFN-β alone vs media, expressed as ratio of IFN-β/media. (B) Vitamin D alone vs media. (C) IFN-β plus vitamin D vs media alone. Selected targets are grouped into Th1, Th17, and Th2 immune pathways and clusters of IFN-stimulated proteins (ISGs), adhesion molecules, and cytokines controlling homeostatic proliferation, neurotrophic factors, and IL-12 family. Values are fold change in the stimulation condition/media. Intensity of shading shows upregulation (red) or downregulation (blue). Target proteins include BDNF = brain-derived neurotrophic factor; HGF = hepatocyte growth factor; IFN-γ = interferon-γ; IL-2 = interleukin-2; TNF-α = tumor necrosis factor-α; TNFRII = TNF receptor type II; IL-17F, IL-4, IL-5, IL-10, IP-10 = IFN-induced protein-10 (CXCL10); MCP1 = macrophage chemotactic protein-1 (CCL2); I-TAC = IFN-inducible T-cell alpha chemoattractant (CXCL11); LIF = leukemia inhibitory factor; p-Y-STAT1 = phosphotyrosine-STAT1 transcription factor; MxA = myxovirus A; NGF = nerve growth factor; sICAM-1 = soluble intercellular adhesion molecule-1; TPO = thymopoietin; VCAM-1 = vascular cell adhesion molecule; VEGF-α = vascular endothelial growth factor-α, IL-15, IL-7, and IL-12 p40 and IL-12 p70 components.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: Derivative Assay

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: (A) In vitro vitamin D enhances IFN-β induction of p-Y-STAT1 in ConA-activated MNCs from 72 untreated patients with MS ( p = 0.00002). (B) p-Y-STAT1 expression on flow cytometry histograms from a representative patient with RRMS-s. The media, IFN, VitD, and IFN+VitD curves are all stained with Alexa Fluor 488–labeled Mab to p-Y-STAT1. (C) MxA protein on Western blot of a representative patient with RRMS-s. ConA-activated MNCs were incubated in vitro with 160 U/mL IFN-β-1b for 30 minutes to 48 hours ± preincubation with 200 nM Vit D3 (calcitriol) for 12 hours. Gray-scale densities of each band are listed above figure. IFN-β = interferon-β; MxA = myxovirus protein.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: In Vitro, Expressing, Flow Cytometry, Staining, Labeling, Western Blot, Incubation

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: (A) p-Y-STAT1, measured with flow cytometry, is generated in all groups, but there is less induction in SPMS than in HC. (B) MxA protein, measured with Western blots. The trend for enhanced responses in PPMS vs HC was not significant ( p < 0.13, unpaired t test). ConA-activated MNCs were incubated in vitro with 160 U/mL IFN-β-1b for 30 minutes to 48 hours ± preincubation with 200 nM vitamin D3 (calcitriol) for 12 hours. Fold change of vitamin D plus IFN-β compared with IFN-β alone, determined using VDSI: [(IFN + VitD) − (VitD)]/[(IFN) − (no IFN)]; average with p values above SEM bar: * p < 0.05, ** p < 0.01. Comparisons between groups use unpaired t tests, brackets. PPMS = primary progressive MS; RRMS-a = active relapsing/remitting MS; RRMS-s = stable RRMS; SPMS = secondary progressive MS.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Generated, Western Blot, Incubation, In Vitro

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: In all groups, there is enhanced induction of p-Y-STAT1, measured with flow cytometry. Methods and calculations as in . * p < 0.05, ** p < 0.01. IFN-β = interferon-β; RRMS-a = active relapsing/remitting MS; RRMS-s = stable RRMS; SPMS = secondary progressive MS.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: Flow Cytometry

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: Vitamin D plus IFN-β–induced p-Y-STAT1 expression is quantitated with flow cytometry. The median vitamin D level for the entire cohort was 30; low < 30, high ≥ 30 ng/mL 25-OH vitamin D. Methods and calculations as in . * p < 0.05, ** p < 0.01. RRMS-a = active relapsing/remitting MS; RRMS-s = stable RRMS; PPMS = primary progressive MS; SPMS = secondary progressive MS.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: Expressing, Flow Cytometry

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: Vitamin D plus IFN-β (blue line) in most therapy-naive groups had a greater effect than IFN-β alone (yellow line) on activation of STAT1 in ConA-activated lymphocytes and monocytes. Values in radar plot represent median fold change in log 2 scale expression of p-Y-STAT1 measured by flow cytometry. Blue line: (IFN+ VitD) − (Vit D); yellow: (IFN)-(media alone). IFN-β = interferon-β; RRMS-a = active relapsing/remitting MS; RRMS-s = stable RRMS; SPMS = secondary progressive MS.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: Activation Assay, Expressing, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: α-Synuclein disrupts microglial autophagy through STAT1-dependent suppression of Ulk1 transcription

doi: 10.1186/s12974-024-03268-4

Figure Lengend Snippet: STAT1 is responsible for the suppression of Ulk1 transcription in α-Syn-treated microglia. (a) Venn diagram demonstrating the number of predicted transcription factors (TFs) of the Ulk1 gene via the hTFtarget, ENCODE, cor_GTEx and KnockTF datasets. A list of potential TFs that regulate Ulk1 transcription is shown in the box. (b , c) qPCR data of Ulk1 mRNA levels (b) and western blot analysis of various protein levels (c) in sh-Vec- or sh-Stat1 (Stat1 KD) -transfected BV2 cells, n = 4. (d) Western blot and quantification of ULK1 and STAT1 protein levels in Stat1 plasmid- or vector - transfected BV2 cells; n = 4. (e) Ulk1 mRNA levels in IFN-γ-treated microglia; n = 3 independent repeats. (f) The binding motif of STAT1 in the promoter of mouse Ulk1 was predicted via JASPAR. The WT and mutated STAT1 regulatory sequences are located 67 bp downstream of the transcription start site (TSS) (+ 1). (g) Dual luciferase assay for Ulk1 promoter activity. HEK293 cells were transfected with pGL3-WT-Ulk1-Luc or pGL3-Mut-Ulk1-Luc , along with pcDNA3.1 or pcDNA3.1-Stat1 , n = 3 independent repeats. (h) ChIP assay for STAT1 enrichment in the Ulk1 promoter. Fold changes are presented, n = 3. (i) Ulk1 mRNA levels in PFF-treated sh-Vec- or sh-Stat1 -transfected BV2 cells, n = 3. (j) Western blot analysis of STAT1 and p-STAT1 Y701 levels in PFF-treated microglia in the presence of TAK-242 (1 µM), n = 4. (k) p-STAT1 Y701 fluorescence and subcellular distribution in PFF-treated WT or Tlr4 −/− microglia. Scale bar, 10 μm. Nuclei were stained with DAPI. The data are presented as the means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant. One-way ANOVA followed by Tukey’s post hoc test was used for i and j , and Student’s t test was used for the other panels

Article Snippet: The primary antibodies used were as follows: anti-ULK1 (1:500, 8054 S, CST, USA), anti-LC3 (1:2000, NB100–2220, Novus, USA), anti-tyrosine hydroxylase (anti-TH, 1:1000, T1299, USA), anti-SQSTM1/p62 (1:1000, P0067, Sigma, USA), anti-p-STAT1 Y701 (1:500, AP0054, ABclonal, China), anti-STAT1 (1:500, 14994, CST, USA), anti-α-Syn (1:500, AB138501, Abcam, USA), anti-ACTB (1:5000, A3854, Sigma, USA) and anti-GAPDH (1:5000, 60004-1-IG, Proteintech, China).

Techniques: Western Blot, Transfection, Plasmid Preparation, Binding Assay, Luciferase, Activity Assay, Fluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: α-Synuclein disrupts microglial autophagy through STAT1-dependent suppression of Ulk1 transcription

doi: 10.1186/s12974-024-03268-4

Figure Lengend Snippet: Microglial STAT1 is activated in α-Syn OE mice. (a , b) Western blots (a) and quantification (b) of p-STAT1 Y701 , ULK1, α-Syn and TH protein levels in the SN of AAV-hα-Syn- or AAV-Vector -injected mice at 8 weeks post injection; n = 4 animals per group. (c-g) Representative images of p-STAT1 Y701 (green) with IBA1 (c) or GFAP (f) costaining in the SNr. The p-STAT1 Y701 intensity (d) and Mander’s overlap coefficient analysis for p-STAT1 Y701 colocalization with IBA1 (e)- or GFAP (g) -positive cells are presented; n = 3 mice per group, with at least three slices per mouse. Aarrowheads indicate p-STAT1 Y701 and IBA1 colocalization, with the enlarged inset on the right. DAPI was used to stain the nuclei. Scale bar, 20 μm. Unpaired Student’s t test. (h , i) Western blot analysis of STAT1 and p-STAT1 Y701 levels in α-Syn PFF-treated microglia (h) and astrocytes (i) ; n = 3–5 replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant. One-way ANOVA followed by Dunnett’s test was used for h and i, and Student’s t test was used for the other panels

Article Snippet: The primary antibodies used were as follows: anti-ULK1 (1:500, 8054 S, CST, USA), anti-LC3 (1:2000, NB100–2220, Novus, USA), anti-tyrosine hydroxylase (anti-TH, 1:1000, T1299, USA), anti-SQSTM1/p62 (1:1000, P0067, Sigma, USA), anti-p-STAT1 Y701 (1:500, AP0054, ABclonal, China), anti-STAT1 (1:500, 14994, CST, USA), anti-α-Syn (1:500, AB138501, Abcam, USA), anti-ACTB (1:5000, A3854, Sigma, USA) and anti-GAPDH (1:5000, 60004-1-IG, Proteintech, China).

Techniques: Western Blot, Plasmid Preparation, Injection, Staining