Journal: Brain, behavior, and immunity

Article Title: Irisin reprograms microglia through activation of STAT6 and prevents cognitive dysfunction after surgery in mice.

doi: 10.1016/j.bbi.2024.12.019

Figure Lengend Snippet: Fig. 9. Schematic representation of the mechanism of irisin in preventing cognitive dysfunction induced by LPS or surgery. ①Exogenous irisin crosses the blood–brain barrier (BBB). ②Irisin directly targets the αVβ5 integrin receptor in microglia and further inhibits the expression of β5 integrin. ③The nuclear transcription factor STAT6 is further activated to promote the polarization of microglia toward the M2 anti-inflammatory phenotype. STAT, signal transducer and activator of transcription. ④Simultaneously, STAT6 inhibits NF-κB activation, and inhibits the polarization to the M1 proinflammatory phenotype induced by LPS or surgery. NF-κB, nuclear factor kappa-B. ⑤Irisin treatment leads to increased expression of anti-inflammatory mediators (namely, IL-10 and Arg-1) and BDNF, and inhibited the expression of proinflammatory factors (namely, IL-1β, IL-18, and TNF-α), thus improving the immune microenvironment. IL, interleukin; Arg-1, arginase-1; BDNF, brain-derived neurotrophic factor. TNF, tumor necrosis factor. ⑥Irisin maintains neuronal homeostasis (including number, dendritic spine density, excitability). ⑦Irisin effectively prevents neurocognitive dysfunction in LPS-induced adult neuroinflammatory mice and surgery/anesthesia-induced early cognitive dysfunction aged mice.

Article Snippet: For staining, the dHPC brain slices were washed 3 times with phosphatebuffered saline (PBS) for 5 min, blocked with a buffer containing 5 % bull serum albumin and 0.3 % Triton X-100 for 1 h, incubated with primary antibodies (1:500), including FNDC5/irisin (ab13190, Abcam), Irisin (H-067–17, Phoenix Pharmaceuticals), Iba1 (ab5076, Abcam), Integrin β5 (3629S, CST), Phospho-NF-κB p65 (3033 T, CST), NF-κB p65 (8242S, CST), STAT6 (5397; CST), Phospho-STAT6 (Tyr641) (56554; CST), c-Fos (2250 s, CST), and NeuN (ab104224, Abcam) at 4 ◦C overnight.

Techniques: Expressing, Activation Assay, Derivative Assay

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: (A) Fluorescence confocal images showing A488-irisin binding in HEK293T cells. HEK293T cells were either transiently transfected with control vector or full-length αV and β5 plasmids. 2 nM Hsp90α was used for 1 hr pretreatment, and 2 nM A488-irisin-His was subsequently used for 5 min treatment. Scale bar: 20 μm. (B) Anti-phosphorylated FAK (Y397) and anti-FAK western blots showing the levels of integrin signaling upon irisin and/or Hsp90α treatments. HEK293T cells were transfected and treated in the same way as (A), except for the addition of the shown amounts of unlabeled irisin-His (0.1 nM or 1 nM) and Hsp90α (1 nM). Anti-αV and anti-β5 antibodies were used to probe the levels of the ectopically expressed αV and β5. (C) Immunofluorescence confocal images showing cell surface Hsp90α in SK-Mel2 cells. Live cells were treated with either control IgG or anti-Hsp90α at 4°C. Scale bar: 50 μm. (D) Quantification of the percentage of SK-Mel2 cells expressing cell surface Hsp90α in (C) (significant if p-value < 0.05 by unpaired t-test). (E) Fluorescence confocal images showing A647-irisin binding in SK-Mel2 cells. Live cells were pretreated with either control IgG or anti-Hsp90α at 4°C for 1 hr followed by 2 nM A647-irisin-His treatment at room temperature for 5 min. Scale bar: 50 μm. (F) Quantification of the percentage of A647-positive cells in (E) (significant if p-value < 0.05 by unpaired t-test). (G) Co-immunoprecipitation assay of endogenous cell surface αV and β5 using SK-Mel2 cells. Endogenous cell surface Hsp90α was captured by anti-Hsp90α in live cells at 4°C. (H) Crystal violet assay showing does-dependent inhibition of the cell viability of SK-Mel2 upon irisin treatment. Grey bar: control treatment with PBS. Concentrations of irisin-His used for the treatments were indicated (one-way ANOVA). (I) Crystal violet assay showing the inhibition of irisin-mediated effect in SK-Mel2 cells by anti-Hsp90α or control antibody. Grey bar: control treatment with PBS. 50 ng/mL of irisin-His was used (one-way ANOVA). (J) Western blot of mouse inguinal fat tissue lysates using the indicated antibodies to probe integrin signaling. Mice were given anti-Hsp90α antibody or control IgG (500 μg/kg) subcutaneously 24 hrs before a bolus injection of recombinant irisin (5 mg/kg) directly into the inguinal fat pads. The mice were sacrificed and inguinal fat tissues were harvested 20 min after irisin injection.

Article Snippet: Rabbit Monoclonal Anti- Integrin β5 , Abcam , Cat. # ab184312.

Techniques: Fluorescence, Binding Assay, Transfection, Control, Plasmid Preparation, Western Blot, Immunofluorescence, Expressing, IF-P, Co-Immunoprecipitation Assay, Crystal Violet Assay, Inhibition, Injection, Recombinant

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: (A) Flow charts of the steps used in three different methods for analyzing αVβ5-Apo and αVβ5/Hsp90α cryo-EM samples. (B) 2D classes (generated by method 1) of αVβ5 particles in each of the three conformational states and the numbers (quantified by all three methods) of particles in each state. (C) Quantification of the percentage of distinguished particles (“likely open” particles were not included) in each of the three conformational states. (D) Fluorescence anisotropy assay for A488-irisin binding by αVβ5, the αVβ5/Hsp90α complex in the presence of 1 mM MgCl2 and 1 mM CaCl2, or αVβ5 in the presence of 1 mM MnCl2. 50 nM A488-irisin-His was used in the assay. (E) Cartoon diagram showing a two-step process of the irisin action through αVβ5. Irisin alone has low affinity for the closed-state αVβ5. Hsp90α, Mn2+ ion, or other possible factors, “opens” αVβ5, allowing for high-affinity irisin binding and effective signaling transduction through its integrin receptor. (F) and (G) TALON pull-downs performed using 1 μM bead-bound clasped and tagged αVβ5. These were mixed with 2 μM untagged Hsp90α without bound nucleotide (Hsp90α-Apo) or Hsp90α charged with the indicated nucleotides (F), or Hsp90α nonhydrolyzing mutant (G95D) (G), and bound samples were analyzed by Coomassie staining and anti-Hsp90α western blot.

Article Snippet: Rabbit Monoclonal Anti- Integrin β5 , Abcam , Cat. # ab184312.

Techniques: Cryo-EM Sample Prep, Generated, Fluorescence, Binding Assay, Transduction, Mutagenesis, Staining, Western Blot

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: Figure 5C

Article Snippet: Rabbit Monoclonal Anti- Integrin β5 , Abcam , Cat. # ab184312.

Techniques: Control, Virus, Recombinant, Expressing, Protease Inhibitor, Lysis, Transfection, Electron Microscopy, Immunodepletion, Clone Assay, Protein Purification, Endotoxin Assay, Bradford Assay, Staining, Mass Spectrometry, Mutagenesis, Software, Isolation