Journal: Research Square

Article Title: Efferocytosis is restricted by axon guidance molecule EphA4 via ERK/Stat6/Mertk signaling following brain injury

doi: 10.21203/rs.3.rs-3079466/v1

Figure Lengend Snippet: (A) Uniform Manifold Approximation and Projection (UMAP) plot showing different cell clusters and their cell-specific annotation in the injured cortex at 1dpi (B) Dot plot of efferocytosis genes expressed by each cluster type. (C-F) Feature maps highlighting the expression across clusters of Mertk (C), Gas 6 (D), Pros1 (E), and Stat6 (F).

Article Snippet: On day 5, bone marrow-derived macrophages (BMDMS) were supplemented with fresh RPMI media or pretreated with 1 μg/ml of Escherichia coli O111:B4 LPS (Sigma Aldrich, St. Louis, MO) for 4 hours, 0.5 μg/ml of mouse recombinant HMGB1 (ThermoFisher Scientific) for 4 hours, 5 μg/ml of EphA4-Fc or Fc-control clustered using 1.7 μg/ml α-Fc (Sino Biological, Wayne, PA) for 1 hour, 25 μM of ERK inhibitor (FR18020R, Cayman chemicals) for 4 hours, 5 μM of Mertk inhibitor (UNC2025, Cayman Chemicals) for 4 hours, or 250 nM of Stat6 inhibitor (AS1517499, Cayman Chemicals) for 4 hours.

Techniques: Expressing

Journal: Research Square

Article Title: Efferocytosis is restricted by axon guidance molecule EphA4 via ERK/Stat6/Mertk signaling following brain injury

doi: 10.21203/rs.3.rs-3079466/v1

Figure Lengend Snippet: Peripheral EphA4 deficiency promotes P-ERK/P-Stat6/Mertk signaling. A-C) mRNA expression of the efferocytosis receptor (Mertk, A) and its ligands (Gas6, B) and (Pros1, C) in the contralateral and ipsilateral cortex of WT +WT BMCs and WT +KO BMCs mice at 3dpi. N=5–6 mice/group. Ns = non-significant; *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001. Two-way ANOVA followed by Šídák’s multiple comparisons test. D) Western blot analysis shows increased Mertk, ERK, and Stat6 phosphorylation in the ipsilateral cortex of chimeric WT +KO BMCs mice. E-H) Representative images showing peripheral-derived GFP+ cells expressing Mertk (purple) and P-ERK (E, F) and P-Stat6 (G, H) in WT +WT BMCs and WT +KO BMCs mice.

Article Snippet: On day 5, bone marrow-derived macrophages (BMDMS) were supplemented with fresh RPMI media or pretreated with 1 μg/ml of Escherichia coli O111:B4 LPS (Sigma Aldrich, St. Louis, MO) for 4 hours, 0.5 μg/ml of mouse recombinant HMGB1 (ThermoFisher Scientific) for 4 hours, 5 μg/ml of EphA4-Fc or Fc-control clustered using 1.7 μg/ml α-Fc (Sino Biological, Wayne, PA) for 1 hour, 25 μM of ERK inhibitor (FR18020R, Cayman chemicals) for 4 hours, 5 μM of Mertk inhibitor (UNC2025, Cayman Chemicals) for 4 hours, or 250 nM of Stat6 inhibitor (AS1517499, Cayman Chemicals) for 4 hours.

Techniques: Expressing, Western Blot, Phospho-proteomics, Derivative Assay

Journal: Research Square

Article Title: Efferocytosis is restricted by axon guidance molecule EphA4 via ERK/Stat6/Mertk signaling following brain injury

doi: 10.21203/rs.3.rs-3079466/v1

Figure Lengend Snippet: EphA4-null BMDMs show enhanced efferocytosis that is regulated by ERK/ Stat6/Mertk. A-I) EphA4 deletion enhances the efferocytosis efficiency of BMDMS in vitro . A-H) Representative images showing the engulfment of the pHrodo-stained apoptotic (but not live) Jurkat cells (red) by GFP+ untreated WT (A-D) and EphA4 KO (E-H) BMDMS. I) Quantification of the efferocytosis efficiency of WT and EphA4 KO BMDMS after stimulation with LPS and HMGB1 for 4 hours. J) Efferocytosis of EphA4 KO BMDMSs is mediated by the blockade of forward EphA4, not reverse ephrin signals. Treatment of WT and EphA4 KO BMDMS with clustered EphA4-FC to activate reverse ephrin signals did not reduce the efferocytosis of EphA4 KO BMDMS. K-M) mRNA expression of Mertk (K), Gas6 (L), and Pros1 (M) with and without engulfment of apoptotic Jurkat cells. EphA4 KO BMDMSs have higher expression of Mertk and Gas6 than WT. N) The use of Mertk inhibitor reduced efferocytosis of both WT and EphA4 KO BMDMS; however, Stat6 and ERK inhibitors selectively reduced efferocytosis in EphA4 KO BMDMS. O) Stat6 inhibitor reduced Mertk and Gas6 expression, and ERK inhibitor reduced Gas6 expression in KO BMDMS engulfing apoptotic Jurkat cells. P) Suggested pathway for the regulation of efferocytosis by EphA4. N=5–6 mice/group. Ns = non-significant; *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001. Two-way ANOVA followed by Šídák’s multiple comparisons test (I-N) or one-way ANOVA followed by Tukey’s multiple comparisons test (O).

Article Snippet: On day 5, bone marrow-derived macrophages (BMDMS) were supplemented with fresh RPMI media or pretreated with 1 μg/ml of Escherichia coli O111:B4 LPS (Sigma Aldrich, St. Louis, MO) for 4 hours, 0.5 μg/ml of mouse recombinant HMGB1 (ThermoFisher Scientific) for 4 hours, 5 μg/ml of EphA4-Fc or Fc-control clustered using 1.7 μg/ml α-Fc (Sino Biological, Wayne, PA) for 1 hour, 25 μM of ERK inhibitor (FR18020R, Cayman chemicals) for 4 hours, 5 μM of Mertk inhibitor (UNC2025, Cayman Chemicals) for 4 hours, or 250 nM of Stat6 inhibitor (AS1517499, Cayman Chemicals) for 4 hours.

Techniques: In Vitro, Staining, Expressing

Journal: Cell reports

Article Title: Early stress-induced impaired microglial pruning of excitatory synapses on immature CRH-expressing neurons provokes aberrant adult stress responses

doi: 10.1016/j.celrep.2022.110600

Figure Lengend Snippet: (A) Representative confocal image of microglia (green; CX3CR1-GFP) abutting CRH + neurons (red; CRH-tdTomato), engulfing vGlut2 + excitatory presynaptic puncta (white; vGlut2 puncta identified as inside microglia by Imaris 3D reconstruction) in mpd PVN of a P8 male mouse. Scale bar, 10 μm (raw images in – ). (B) Representative electron micrograph of a microglia labeled with ionized calcium binding adaptor molecule (Iba)1 and 3′,3-diaminobenzidine (DAB; electron-dense soma and processes) in P8 PVN. The yellow arrowhead points to the postsynaptic density of an excitatory synapse directly abutting a labeled microglial process. The black arrowhead indicates multiple lysosomes (electron dense) in microglial soma; these degrade engulfed material. The blue arrowhead points to an engulfed endosome of synaptic elements, including a putative postsynaptic density, surrounded by lysosomes in a microglia process. Scale bar, 5 μm. (C) ELA reduces the number of vGlut2 + synaptic puncta engulfed by microglia abutting CRH + neurons in the P8 mpd PVN of male mice (t 13.87 = 2.22, p = 0.04; Welch’s t test; see for female data), as quantified in confocal 3D reconstructions. (D) Representative confocal image of the microglial phagocytic receptor Mer tyrosine kinase (MerTK) expression. MerTK is primarily in microglia in the P8 PVN (white ROI; see also ). Red = CRH-tdTomato + neurons; green = MerTK immunoreactivity (IR); magenta = P2RY12 IR (microglia); white = overlap of green and magenta in composite image. Scale bar, 50 μm. (E) Representative confocal images of MerTK IR in CTL (top) compared to ELA (bottom) P8 PVN (white ROI). Scale bar, 50 μm. (F) MerTK volume per unit volume of P2RY12 + microglia (measured using Imaris) was lower in P8 ELA male PVN than in controls (t 6.46 = 3.44, p = 0.01; Welch’s t test). (G) Notably, P2RY12 volume and its percentage of PVN volume did not differ in ELA compared to control mice. (H) The ratio of MerTK mean fluorescence intensity (MFI; measured using Imaris) to P2RY12 MFI was significantly reduced in ELA mice PVN (t 9 = 2.54, p = 0.03; unpaired t test). (I) Schematic of the MerTK inhibition experiment: Litters of CRH-tdTomato + mice were randomly assigned to CTL or ELA conditions on P2. On P6–7, organotypic hypothalamic slice cultures were prepared and maintained for 7 d in vitro (DIV). On DIV7, cultures were treated with 20 nM of a small-molecule MerTK inhibitor (UNC2025: ~40-fold greater selectivity for MerTK over the Axl and Tyro3 TAM receptors [ ; ]) or vehicle. Twelve hours later, the medium was refreshed with new drug, and the cultures were fixed 4 h later . (J) MerTK inhibition increased the number of excitatory synapses on CRH + neurons in PVN cultures from control mice but not in ELA mice (significant interaction of ELA × drug, F 1,22 = 17.89, p = 0.0003; 2-way ANOVA; p < 0.05; post hoc Tukey’s test). Means ± SEMs; *p < 0.05.

Article Snippet: 6 days after initial culture, each membrane was washed twice with 2 mL of antibiotic-free, serum-free medium (97% MEM supplemented with 3 mM L-Glutamine, 10 mM D-Glucose,1.9 mM NaHCO3, 12.5 mM HEPES, 0.6 mM L-Ascorbic acid, 1 μg/mL Insulin) and treatment with 20 nM of a small-molecule MerTK inhibitor as previously described (UNC2025, Selleck Chemicals, cat# S7576) ( ) or sterile culture-grade water as vehicle was commenced.

Techniques: Labeling, Binding Assay, Expressing, Control, Fluorescence, Inhibition, In Vitro

Journal: Cell reports

Article Title: Early stress-induced impaired microglial pruning of excitatory synapses on immature CRH-expressing neurons provokes aberrant adult stress responses

doi: 10.1016/j.celrep.2022.110600

Figure Lengend Snippet:

Article Snippet: 6 days after initial culture, each membrane was washed twice with 2 mL of antibiotic-free, serum-free medium (97% MEM supplemented with 3 mM L-Glutamine, 10 mM D-Glucose,1.9 mM NaHCO3, 12.5 mM HEPES, 0.6 mM L-Ascorbic acid, 1 μg/mL Insulin) and treatment with 20 nM of a small-molecule MerTK inhibitor as previously described (UNC2025, Selleck Chemicals, cat# S7576) ( ) or sterile culture-grade water as vehicle was commenced.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Software, Microscopy, Imaging

Journal: Cell reports

Article Title: Early stress-induced impaired microglial pruning of excitatory synapses on immature CRH-expressing neurons provokes aberrant adult stress responses

doi: 10.1016/j.celrep.2022.110600

Figure Lengend Snippet: (A) Representative confocal image of microglia (green; CX3CR1-GFP) abutting CRH + neurons (red; CRH-tdTomato), engulfing vGlut2 + excitatory presynaptic puncta (white; vGlut2 puncta identified as inside microglia by Imaris 3D reconstruction) in mpd PVN of a P8 male mouse. Scale bar, 10 μm (raw images in – ). (B) Representative electron micrograph of a microglia labeled with ionized calcium binding adaptor molecule (Iba)1 and 3′,3-diaminobenzidine (DAB; electron-dense soma and processes) in P8 PVN. The yellow arrowhead points to the postsynaptic density of an excitatory synapse directly abutting a labeled microglial process. The black arrowhead indicates multiple lysosomes (electron dense) in microglial soma; these degrade engulfed material. The blue arrowhead points to an engulfed endosome of synaptic elements, including a putative postsynaptic density, surrounded by lysosomes in a microglia process. Scale bar, 5 μm. (C) ELA reduces the number of vGlut2 + synaptic puncta engulfed by microglia abutting CRH + neurons in the P8 mpd PVN of male mice (t 13.87 = 2.22, p = 0.04; Welch’s t test; see for female data), as quantified in confocal 3D reconstructions. (D) Representative confocal image of the microglial phagocytic receptor Mer tyrosine kinase (MerTK) expression. MerTK is primarily in microglia in the P8 PVN (white ROI; see also ). Red = CRH-tdTomato + neurons; green = MerTK immunoreactivity (IR); magenta = P2RY12 IR (microglia); white = overlap of green and magenta in composite image. Scale bar, 50 μm. (E) Representative confocal images of MerTK IR in CTL (top) compared to ELA (bottom) P8 PVN (white ROI). Scale bar, 50 μm. (F) MerTK volume per unit volume of P2RY12 + microglia (measured using Imaris) was lower in P8 ELA male PVN than in controls (t 6.46 = 3.44, p = 0.01; Welch’s t test). (G) Notably, P2RY12 volume and its percentage of PVN volume did not differ in ELA compared to control mice. (H) The ratio of MerTK mean fluorescence intensity (MFI; measured using Imaris) to P2RY12 MFI was significantly reduced in ELA mice PVN (t 9 = 2.54, p = 0.03; unpaired t test). (I) Schematic of the MerTK inhibition experiment: Litters of CRH-tdTomato + mice were randomly assigned to CTL or ELA conditions on P2. On P6–7, organotypic hypothalamic slice cultures were prepared and maintained for 7 d in vitro (DIV). On DIV7, cultures were treated with 20 nM of a small-molecule MerTK inhibitor (UNC2025: ~40-fold greater selectivity for MerTK over the Axl and Tyro3 TAM receptors [ ; ]) or vehicle. Twelve hours later, the medium was refreshed with new drug, and the cultures were fixed 4 h later . (J) MerTK inhibition increased the number of excitatory synapses on CRH + neurons in PVN cultures from control mice but not in ELA mice (significant interaction of ELA × drug, F 1,22 = 17.89, p = 0.0003; 2-way ANOVA; p < 0.05; post hoc Tukey’s test). Means ± SEMs; *p < 0.05.

Article Snippet: MerTK small-molecule inhibitor, UNC2025 , Selleck Chemicals , Cat# S7576.

Techniques: Labeling, Binding Assay, Expressing, Control, Fluorescence, Inhibition, In Vitro

Journal: Cell reports

Article Title: Early stress-induced impaired microglial pruning of excitatory synapses on immature CRH-expressing neurons provokes aberrant adult stress responses

doi: 10.1016/j.celrep.2022.110600

Figure Lengend Snippet:

Article Snippet: MerTK small-molecule inhibitor, UNC2025 , Selleck Chemicals , Cat# S7576.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Software, Microscopy, Imaging