Journal: Journal of Neuroinflammation

Article Title: Soluble CSF1R promotes microglial activation and amyloid clearance in alzheimer’s disease

doi: 10.1186/s12974-025-03558-5

Figure Lengend Snippet: Elevated sCSF1R levels in the CSF of Alzheimer’s disease patients across multiple independent cohorts. a-b Mass spectrometry-based proteomic analysis of CSF samples from two independent cohorts reported by Ali et al. revealed significantly increased levels of sCSF1R in A + T + AD patients compared to A − T − controls. c-d Independent validation using a dataset from Dammer et al. confirmed that sCSF1R levels in the CSF were elevated in AD patients compared with controls. sCSF1R protein abundance was quantified using tandem mass tag-based mass spectrometry (TMT-MS) ( c ) and the SomaScan platform ( d ). e-h Correlation analyses between the sCSF1R in CSF (measured by TMT-MS) and AD-related biomarkers, including total tau (tTau; e ), phosphorylated tau at threonine 181 (pTau181; f ), β-amyloid 1–42 (Aβ42; g ), and cognitive performance assessed by the Montreal Cognitive Assessment (MoCA) score ( h ) (Pearson correlation coefficient test). i-l Correlation analyses between the sCSF1R in CSF (measured by SomaScan) and tTau ( i ), pTau181 ( j ), Aβ42 ( k ), and MoCA score ( l ) (Pearson correlation coefficient test). ** p < 0.01; **** p < 0.0001

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST and incubated overnight at 4 °C with the following primary antibodies: CSF1R (Abcam, ab37858, 1:2000), sCSF1R (Sino Biological, 50059-RP01, 1:2000), Iba1 (Cell Signaling Technology, 17198 S, 1:1000), β-actin (Cell Signaling Technology, 4970, 1:10000), ADAM17 (Cell Signaling Technology, 61048, 1:2000).

Techniques: Mass Spectrometry, Biomarker Discovery, Quantitative Proteomics

Journal: Journal of Neuroinflammation

Article Title: Soluble CSF1R promotes microglial activation and amyloid clearance in alzheimer’s disease

doi: 10.1186/s12974-025-03558-5

Figure Lengend Snippet: Pathological microglial activation induces sCSF1R elevation in vivo and in vitro. a-b Representative images ( a ) and quantification ( b ) of nuclei (blue) and Iba1 (red) immunostaining in the hippocampus of seven- to eight-month-old wild-type (WT) mice, LPS-injected WT mice, and 5×FAD mice ( n = 6–8 mice per group; unpaired Student’s t -test). Scale bars, 150 μm. c-e Representative images ( c ) and quantification ( d-e ) of nuclei (blue), Iba1 (red), CD68 (green), and Clec7a (yellow) immunostaining in the hippocampus of seven- to eight-month-old WT mice, LPS-injected WT mice, and 5×FAD mice ( n = 6–8 mice per group; unpaired Student’s t -test). Scale bars, 25 μm. f ELISA-based quantification of sCSF1R in the CSF from seven- to eight-month-old WT mice, LPS-injected WT mice, and 5×FAD mice ( n = 6 mice per group; one-way ANOVA). g-i Western blot analysis of full-length CSF1R, shed sCSF1R, and Iba1 in the hippocampal lysates from seven- to eight-month-old WT mice, LPS-injected WT mice, and 5×FAD mice ( n = 6–8 mice per group; unpaired Student’s t -test). j-l Western blot analysis of full-length CSF1R in cell lysates and sCSF1R in culture media from primary microglia after 3-hour treatment with 10 µM oligomeric Aβ42 (oAβ) and fibrillar Aβ42 (fAβ) ( n = 6–8; from three independent experiments; unpaired Student’s t -test). m-o Western blot analysis of full-length CSF1R in cell lysates and sCSF1R in culture media from primary microglia treated with 10 ng/mL LPS or 25 ng/mL GM-CSF ( n = 6; from three independent experiments; one-way ANOVA). Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST and incubated overnight at 4 °C with the following primary antibodies: CSF1R (Abcam, ab37858, 1:2000), sCSF1R (Sino Biological, 50059-RP01, 1:2000), Iba1 (Cell Signaling Technology, 17198 S, 1:1000), β-actin (Cell Signaling Technology, 4970, 1:10000), ADAM17 (Cell Signaling Technology, 61048, 1:2000).

Techniques: Activation Assay, In Vivo, In Vitro, Immunostaining, Injection, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Soluble CSF1R promotes microglial activation and amyloid clearance in alzheimer’s disease

doi: 10.1186/s12974-025-03558-5

Figure Lengend Snippet: sTREM2 promotes sCSF1R production via ADAM17-mediated cleavage. a-c Western blot analysis of full-length CSF1R in cell lysates and sCSF1R in culture media from primary microglia treated with 40 nM sTREM2 for various time points, with or without the lysosomal inhibitor chloroquine (CQ, 30 µM) or the proteasome inhibitor MG132 (1 µM) ( n = 5 independent experiments; two-way ANOVA). d-f Western blot analysis of full-length CSF1R in cell lysates and sCSF1R in culture media from primary microglia after 1-hour treatment with 40 nM sTREM2 in the presence or absence of 30 µM ADAM17 inhibitor TAPI-1 ( n = 7; from three independent experiments; two-way ANOVA). g-i Western blot analysis of full-length CSF1R in cell lysates and sCSF1R in culture media from primary microglia after 1-hour treatment with 40 nM sTREM2 in the presence or absence of 10 µM ADAM17 specific inhibitor DPC 333 ( n = 6; from three independent experiments; two-way ANOVA). j Relative mRNA levels of ADAM17 in microglia treated with 40 nM sTREM2 for 5 h, measured by RT-PCR and normalized to β-actin ( n = 7; from three independent experiments; unpaired Student’s t -test). k Western blot analysis of ADAM17 in microglia treated with 40 nM sTREM2 for 1 h ( n = 7; from three independent experiments; unpaired Student’s t -test). l Enzymatic activity of ADAM17 in microglia treated with 40 nM sTREM2 for 1 h ( n = 8; from four independent experiments; unpaired Student’s t -test). m-n Representative images ( m ) and quantification ( n ) of nuclei (blue) and Iba1 (red) immunostaining in the hippocampus of seven-month-old wild-type mice injected with sTREM2 protein or vehicle control for three days ( n = 3 mice per group; paired Student’s t -test). Scale bars, 150 μm. o-p Relative mRNA levels of microglial homeostatic genes in the hippocampal tissue lysates from sTREM2-injected wild-type mice, measured by RT-PCR and normalized to β-actin ( n = 3 mice per group; paired Student’s t -test). q-s Relative mRNA levels of disease-associated microglial and inflammation-related genes in the hippocampal tissue lysates from sTREM2-injected wild-type mice, normalized to β-actin ( n = 3 mice per group; paired Student’s t -test). t-v Western blot analysis ( t ) and quantification ( u-v ) of full-length CSF1R, shed sCSF1R, and Iba1 in the hippocampus of sTREM2-injected wild-type mice ( n = 3 mice per group; paired Student’s t -test). Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST and incubated overnight at 4 °C with the following primary antibodies: CSF1R (Abcam, ab37858, 1:2000), sCSF1R (Sino Biological, 50059-RP01, 1:2000), Iba1 (Cell Signaling Technology, 17198 S, 1:1000), β-actin (Cell Signaling Technology, 4970, 1:10000), ADAM17 (Cell Signaling Technology, 61048, 1:2000).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Immunostaining, Injection, Control

Journal: Journal of Neuroinflammation

Article Title: Soluble CSF1R promotes microglial activation and amyloid clearance in alzheimer’s disease

doi: 10.1186/s12974-025-03558-5

Figure Lengend Snippet: sCSF1R enhances microglial survival, migration, phagocytosis, and inflammatory response. a Silver staining analysis of purified human sCSF1R protein. b-e Relative mRNA levels of homeostatic and disease-associated microglial genes in microglia treated with 40 nM sCSF1R for 5 h, measured by RT-PCR and normalized to β-actin ( n = 7–9; from three independent experiments; unpaired Student’s t -test). f-h Quantification of inflammatory-related genes expression in microglia after 5-hour treatment with 40 nM sCSF1R, assessed by RT-PCR with β-actin as internal control ( n = 9; from three independent experiments; unpaired Student’s t -test). i-j Representative images ( i ) and quantification ( j ) of microglial migration following 24-hour treatment with 40 nM sCSF1R. Migrated cells stained with hematoxylin and eosin were imaged using a Nikon inverted microscope ( n = 8; from three independent experiments; unpaired Student’s t -test). Scale bars, 400 μm. k Microglial survival analysis after 24-hour treatment with 40 nM sCSF1R, determined by CCK-8 assay ( n = 4 independent experiments; unpaired Student’s t -test). l-m Representative images ( l ) and quantification ( m ) of microglial nuclei (blue) and apoptotic cells (green) after 24-hour treatment with 40 nM sCSF1R, assessed using a Colorimetric TUNEL Apoptosis Assay Kit ( n = 9; from three independent experiments; unpaired Student’s t -test). Scale bars, 150 μm. n-o Evaluation of microglial phagocytosis by immunofluorescence following treatment with 40 nM sCSF1R for 24 h and 200 nM FAM-Aβ42 for 3 h ( n = 8–9; from four independent experiments; unpaired Student’s t -test). Scale bars, 50 μm. p-q Flow cytometry analysis of microglial phagocytic capacity following the same treatments as those shown in panel ( n ) ( n = 9; from three independent experiments; unpaired Student’s t -test). All data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST and incubated overnight at 4 °C with the following primary antibodies: CSF1R (Abcam, ab37858, 1:2000), sCSF1R (Sino Biological, 50059-RP01, 1:2000), Iba1 (Cell Signaling Technology, 17198 S, 1:1000), β-actin (Cell Signaling Technology, 4970, 1:10000), ADAM17 (Cell Signaling Technology, 61048, 1:2000).

Techniques: Migration, Silver Staining, Purification, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Staining, Inverted Microscopy, CCK-8 Assay, TUNEL Assay, Apoptosis Assay, Immunofluorescence, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: Soluble CSF1R promotes microglial activation and amyloid clearance in alzheimer’s disease

doi: 10.1186/s12974-025-03558-5

Figure Lengend Snippet: sCSF1R activates microglia in vivo. a Schematic diagram of the experimental design. b-d Seven-month-old wild-type mice received stereotaxic injections of 5 µg purified sCSF1R protein into the right hippocampus and heat-inactivated sCSF1R protein (Ctrl) into the contralateral hippocampus for seven days. Coronal brain sections were stained with DAPI (blue) to visualize nuclei and Iba1 (red) to label microglia ( b ). Quantification of microglial coverage ( c ) and microglial cell number ( d ) was performed ( n = 5 mice per group; paired Student’s t -test). Scale bars, 150 μm. e-f Microglial branching was quantified in sCSF1R-injected wild-type mice ( n = 5 mice per group; two-way ANOVA). Scale bars, 15 μm. g-i The activated microglial phenotype was assessed by CD68 staining (white). Quantification of the percentage of CD68-positive microglial area ( h ) and CD68 content per microglia ( i ) was conducted ( n = 5 mice per group; paired Student’s t -test). Scale bars, 15 μm. j-l Coronal sections were stained with DAPI (blue), Iba1 (red), the homeostatic microglial marker P2ry12 (green), and the disease-associated microglial marker Clec7a (white). Quantification of P2ry12 ( k ) and Clec7a ( l ) coverage was performed ( n = 5 mice per group; paired Student’s t -test). Scale bars, 50 μm. All data are presented as mean ± SEM. ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST and incubated overnight at 4 °C with the following primary antibodies: CSF1R (Abcam, ab37858, 1:2000), sCSF1R (Sino Biological, 50059-RP01, 1:2000), Iba1 (Cell Signaling Technology, 17198 S, 1:1000), β-actin (Cell Signaling Technology, 4970, 1:10000), ADAM17 (Cell Signaling Technology, 61048, 1:2000).

Techniques: In Vivo, Purification, Staining, Injection, Marker

Journal: Journal of Neuroinflammation

Article Title: Soluble CSF1R promotes microglial activation and amyloid clearance in alzheimer’s disease

doi: 10.1186/s12974-025-03558-5

Figure Lengend Snippet: sCSF1R ameliorates amyloid plaque pathology in 5×FAD mice. a-b Seven-month-old 5×FAD mice received stereotaxic injections of 5 µg purified sCSF1R protein into the right hippocampus and heat-inactivated sCSF1R protein (Ctrl) into the contralateral hippocampus for seven days. Coronal brain sections were stained with DAPI (blue) for nuclei, Iba1 (red) for microglia, and MOAB-2 (green) for Aβ plaques ( a ). Quantification of Aβ plaques coverage across the brain is shown in ( b ) ( n = 8 mice per group; paired Student’s t -test). Scale bars, 700 μm. c Representative hippocampal images showing Aβ plaques (MOAB-2, green), microglia (Iba1, red), and nuclei (DAPI, blue) from 5×FAD mice treated with sCSF1R. d-f Quantification of amyloid pathology in the hippocampus, including plaque coverage ( d ), plaque number ( e ), and microglial coverage ( f ) ( n = 9 mice per group; paired Student’s t -test). Scale bars, 100 μm. g High-magnification Z-stack projections showing Aβ plaques (MOAB-2, green) and microglia (Iba1, red) in the hippocampus of sCSF1R-treated 5×FAD mice. Scale bars, 15 μm. h Quantification of plaque-associated microglia in ( g ) ( n = 9 mice per group, paired Student’s t -test). i Immunofluorescence images of hippocampal sections from sCSF1R-treated 5×FAD mice stained for Aβ plaques (MOAB-2, green), microglia (Iba1, red), CD68 (white), and nuclei (DAPI, blue), showing microglial phagocytic activity. Three-dimensional reconstructions illustrate Aβ, Iba1-CD68 colocalization, and Iba1-CD68-Aβ colocalization. Scale bars, 15 μm. j Quantification of Iba1-CD68-Aβ colocalization within plaques ( n = 8 mice per group, paired Student’s t -test). k-l Confocal images of Thioflavin-S-stained amyloid deposits (white), showing three distinct plaque subtypes ( k ). Scale bars, 20 μm. The relative fraction of each plaque subtype was quantified ( l ) ( n = 8 mice per group; paired Student’s t -test). m Immunofluorescence images showing Aβ plaques (MOAB-2, green) and dystrophic neurites (LAMP1, white) in the hippocampus of sCSF1R-treated 5×FAD mice. Scale bars, 100 μm/15 µm. n Quantification of LAMP1-positive dystrophic neurite area per plaque ( n = 9 mice per group, paired Student’s t -test). All data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST and incubated overnight at 4 °C with the following primary antibodies: CSF1R (Abcam, ab37858, 1:2000), sCSF1R (Sino Biological, 50059-RP01, 1:2000), Iba1 (Cell Signaling Technology, 17198 S, 1:1000), β-actin (Cell Signaling Technology, 4970, 1:10000), ADAM17 (Cell Signaling Technology, 61048, 1:2000).

Techniques: Purification, Staining, Immunofluorescence, Activity Assay

Journal: Journal of Neuroinflammation

Article Title: Soluble CSF1R promotes microglial activation and amyloid clearance in alzheimer’s disease

doi: 10.1186/s12974-025-03558-5

Figure Lengend Snippet: Schematic diagram illustrating the role of sCSF1R in modulating microglial function and Aβ pathology. Upon activation by various stimuli, membrane-bound CSF1R on microglia undergoes proteolytic cleavage by ADAM17, leading to the release of soluble CSF1R (sCSF1R). Once generated, sCSF1R acts in an autocrine manner to further enhance microglial activation, promoting cell survival, chemotaxis, pro-inflammatory signaling, and Aβ phagocytosis. This forms a positive feedback loop that amplifies microglial responses. In 5×FAD mice, elevating sCSF1R levels enhances microglial recruitment and clustering around plaques, reduces Aβ burden, and alleviates neuritic dystrophy. Image created with BioRender.com

Article Snippet: Membranes were blocked with 5% non-fat milk in PBST and incubated overnight at 4 °C with the following primary antibodies: CSF1R (Abcam, ab37858, 1:2000), sCSF1R (Sino Biological, 50059-RP01, 1:2000), Iba1 (Cell Signaling Technology, 17198 S, 1:1000), β-actin (Cell Signaling Technology, 4970, 1:10000), ADAM17 (Cell Signaling Technology, 61048, 1:2000).

Techniques: Activation Assay, Membrane, Generated, Chemotaxis Assay