Journal: bioRxiv

Article Title: Inhibition of IL34 unveils tissue-selectivity and is sufficient to reduce microglial proliferation in chronic neurodegeneration

doi: 10.1101/2020.03.09.976118

Figure Lengend Snippet: Activation of the CSF1R signalling pathway by IL-34 and CSF-1 and inhibition of IL-34-mediated cell growth using IL-34 neutralizing antibodies. [A] N13 microglia cell line was stimulated with IL-34 (50 or 100 ng/mL), CSF-1 (50 or 100 ng/mL) or LPS (1 μg/mL) for 5 or 10 minutes. Cell lysates were subjected to Western blotting which showed increased phosphorylation of CSF1R and downstream ERK1/2 and AKT after IL-34 and CSF-1 stimulation. [B] IL-34 neutralizing antibodies used in this study (mouse monoclonal IgG2A (v1.1, Ma et al., 2012), rat monoclonal IgG2A (MAB5195, R&D Systems) and sheep polyclonal IgG (AF5195, R&D Systems)) prevented IL-34-dependent cell growth of M-NFS-60 in a similar nanomolar range. n=3, data shown represent mean ± SEM, two-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In white clear bottom 96-well plates 10 μL IL-34 antibody (mouse monoclonal IgG2A (v1.1 manufactured by Genscript, Ma et al., 2012), rat monoclonal IgG2A (MAB5195, R&D Systems) and sheep polyclonal IgG (AF5195, R&D Systems) and 10 μL IL-34 stimulus (R&D systems, 5195-ML-CF) were incubated at 37°C for 30 minutes before 80 μL M-NFS-60 cells (10 3 cells/well) were added.

Techniques: Activation Assay, Inhibition, Western Blot, Phospho-proteomics, Comparison

Journal: bioRxiv

Article Title: Inhibition of IL34 unveils tissue-selectivity and is sufficient to reduce microglial proliferation in chronic neurodegeneration

doi: 10.1101/2020.03.09.976118

Figure Lengend Snippet: Intracerebral IL-34 antibody administration resulted in reduced microglia proliferation in ME7 prion mice. [A] Mice infected with prion disease received a single intracerebral injection of mouse- or human-specific anti-IL-34 (sheep polyclonal IgG) and brains were analysed one week later. [B] Histological analysis of BrdU/Iba1-positive microglial cells in the cortex showed a reduction after treatment with a mouse-, but not with a human-specific antibody. Scale bar 100 μm. Naïve n=6, ME7 n=12, ME7 + isotype n=10, ME7 + anti-mIL-34 n=8, ME7 + anti-hIL-34 n=7, data shown represent mean ± SEM, two-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In white clear bottom 96-well plates 10 μL IL-34 antibody (mouse monoclonal IgG2A (v1.1 manufactured by Genscript, Ma et al., 2012), rat monoclonal IgG2A (MAB5195, R&D Systems) and sheep polyclonal IgG (AF5195, R&D Systems) and 10 μL IL-34 stimulus (R&D systems, 5195-ML-CF) were incubated at 37°C for 30 minutes before 80 μL M-NFS-60 cells (10 3 cells/well) were added.

Techniques: Infection, Injection, Comparison

Journal: Development (Cambridge, England)

Article Title: CSF1R ligands promote microglial proliferation but are not the sole regulators of developmental microglial proliferation

doi: 10.1242/dev.204610

Figure Lengend Snippet: CSF1R ligands boost microglial proliferation in serum-free primary microglial culture. (A) Schematic of immunopanning for the isolation and culture of murine microglia. I. V., in vitro . Schematic created in BioRender by Ho, M. 2025. https://BioRender.com/zvx72w4 . This figure was sublicensed under CC-BY 4.0 terms. (B) A plot of the percentage of total Cd11b + microglia that were KI67 + after a 72-h treatment of 10 ng/ml of each predicted signalling factor in microglial base media. (C) Representative images of proliferative microglia (CD11b + KI67 + ) at 1000 ng/ml. Scale bars: 100 µm. (D) A plot of the mean percentage of microglia that are proliferative at tenfold increasing concentrations of CSF1 or IL34. (E) A plot of the mean percentage of viable microglial sustained by tenfold increasing concentrations of CSF1 or IL34. BM here indicates a microglial base media control supplemented with 2 ng/ml TGFβ2; GM indicates microglial growth media control containing both 2 ng/ml TGFβ2 and 10 ng/ml CSF1. (F) A plot of the percentage of live microglia sustained by BM containing either 1 ng/ml or 10 ng/ml CSF1. Bars represent mean±s.e.m.; B: n =2, D-F: n =3, independent microglial cultures with treatments in triplicate or quadruplicate. **** P <0.0001 (one-way ANOVA, Tukey post-hoc). ns, not significant.

Article Snippet: For dose response experiments, we created tenfold dilutions of CSF1 (Gibco, 315-02) and IL34 (R&D Systems, 5195-ML) in microglial growth media.

Techniques: Isolation, In Vitro, Control

Journal: Development (Cambridge, England)

Article Title: CSF1R ligands promote microglial proliferation but are not the sole regulators of developmental microglial proliferation

doi: 10.1242/dev.204610

Figure Lengend Snippet: CSF1 and IL34 protein increase throughout development and display distinct spatiotemporal transcriptional expression patterns. (A,B) IL34 (A) and CSF1 (B) protein levels in whole-brain homogenates as measured by ELISA. (C) Representative brain microscopy image with regions of interest outlined in red. Scale bar: 100 µm. (D,F) Representative images of Il34 transcript visualized with RNAscope™ in CA1 (D) and somatosensory cortex (F). Scale bars: 40 µm. (E,G) Plots of the mean percentage of total cells of CA1 (E) and somatosensory cortex (G) that are Il34 + across development. (H,J) Representative images of Csf1 transcript visualized with RNAscope™ in CA1 (H) and somatosensory cortex (J). Scale bars: 40 µm. (I,K) Plots of the mean percentage of total cells of CA1 (I) and somatosensory cortex (K) that are Csf1 + across development. (L) Density maps depicting the spatiotemporal expression patterns of Il34 and Csf1 transcripts in RNAscope™ across development. Warmer colours indicate greater, while cooler colours indicate lower, transcript density. Bars represent mean±s.e.m.; A,B: n =5 mice per time point; E: n =3-5 mice per time point, G: n =4-5 mice per time point, I,K: n =2-3 mice per time point. * P <0.05, **** P <0.0001 (one-way ANOVA, Tukey post-hoc).

Article Snippet: For dose response experiments, we created tenfold dilutions of CSF1 (Gibco, 315-02) and IL34 (R&D Systems, 5195-ML) in microglial growth media.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Microscopy, RNAscope

Journal: Development (Cambridge, England)

Article Title: CSF1R ligands promote microglial proliferation but are not the sole regulators of developmental microglial proliferation

doi: 10.1242/dev.204610

Figure Lengend Snippet: CSF1R signalling is largely restricted to microglia and border-associated macrophages in the P7 hippocampus. (A) UMAP plot depicting the cellular populations of the P7 hippocampus. Dots represent individual cells clustered based on transcriptional similarity, while colours indicate distinct cell lineages or cellular states based on gene expression patterns. (B) UMAP plot depicting expression of Csf1r in the P7 hippocampus. (C) UMAP plot depicting expression of Csf1 in the P7 hippocampus. (D) UMAP plot depicting expression of Il34 in the P7 hippocampus. (E) Dot plot depicting the cell population expression levels of Csf1r , Csf1 and Il34 in the P7 hippocampus. (F) CellChat plot predicting the dominant sender populations that secrete CSF1 and IL34 and dominant receiver populations that receive signalling via CSF1R in the P7 hippocampus. (G) Violin plot depicting the expression of microglial Csf1r across the mouse lifespan. (H) UMAP plot depicting inhibitory neurons, excitatory neurons and non-neuronal cells of the adult hippocampus. Dots represent individual cells clustered based on transcriptional similarity, while colours indicate distinct cell lineages or cellular states based on gene expression patterns. (I) UMAP plot depicting expression of Il34 in the adult hippocampus. (J) Violin plot comparing the expression level of Il34 between inhibitory neurons, excitatory neurons and non-neuronal cells of the adult hippocampus. Datasets for A-F: ; G: ; H-J: . G,J: **** P <0.0001 (one-way ANOVA, Tukey post-hoc).

Article Snippet: For dose response experiments, we created tenfold dilutions of CSF1 (Gibco, 315-02) and IL34 (R&D Systems, 5195-ML) in microglial growth media.

Techniques: Gene Expression, Expressing

Journal: Development (Cambridge, England)

Article Title: CSF1R ligands promote microglial proliferation but are not the sole regulators of developmental microglial proliferation

doi: 10.1242/dev.204610

Figure Lengend Snippet: Unaltered Il34 and Csf1 levels of IL1α and IL1β knockout mice are insufficient to explain aberrant developmental microglial proliferation. (A) Representative whole brain microscopy image with analysed regions of interest outlined in red. (B) Representative images of IBA1 + microglia (red) and KI67 + proliferative cells (white) in the P10 hippocampus and cortex of IL1α and IL1β knockout mice. Scale bars: 40 µm. (C,D) Plots of the mean percentage of hippocampal microglia (C) and somatosensory cortex microglia (D) that are proliferating in wild-type (WT), IL1α and IL1β knockout mice across development. (E,F) Plots of mean hippocampal microglial densities (E) and mean somatosensory cortex microglial densities (F) in WT, IL1α and IL1β knockout mice. (G,H) Plots of the mean percentage of total cells of CA1 that are Il34 + (G) or Csf1 + (H) at P10 in WT, IL1α and IL1β knockout mice. (I,J) Plots of the mean percentage of total cells of somatosensory cortex that are Il34 + (I) or Csf1 + (J) at P10 in WT, IL1α and IL1β knockout mice. Bars represent mean±s.e.m.; C-F: n =6-8 mice per time point; G,I: n =5 mice per genotype; H,J: n =3-5 mice per genotype. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 (C-F: two-way ANOVA, Tukey post-hoc; G-J: one-way ANOVA, Tukey post-hoc).

Article Snippet: For dose response experiments, we created tenfold dilutions of CSF1 (Gibco, 315-02) and IL34 (R&D Systems, 5195-ML) in microglial growth media.

Techniques: Knock-Out, Microscopy

Journal: Nature Communications

Article Title: CNS-wide repopulation by hematopoietic-derived microglia-like cells corrects progranulin deficiency in mice

doi: 10.1038/s41467-024-49908-4

Figure Lengend Snippet: a Experimental timeline showing sampling time points after BU or BU + PLX and CAG-GFP BMT in C57BL/6 mice. b – c Flow cytometry analyses. b Fraction of CD45 + CD11b+ cells/total CD45+ cells measured in the brain of mice at the indicated time points ( a ). The quantification and statistics are relative to untreated mice (* p -value vs U). Asterisks: p-value BU vs U (light blue), p-value BU + PLX vs U (dark blue). c Fraction of GFP+ microglia-like cells (MGLCs) in the brain at the indicated time points ( a ). The statistics compare BU vs BU + PLX. b – c U n = 3; BU day 14 n = 3, day 40 n = 4; BU + PLX n = 3 from day 21 to day 40. c The data points for days 90 and 270 are plotted from the experiments described in Fig. b and , respectively: BU day 90 n = 4, day 270 n = 10; BU + PLX day 90 n = 4, day 270 n = 7. d – g Serial cytokine analysis performed on whole brain lysates by 48-plex Luminex or ELISA assays (IL34 and SDF-1/CXCL12) at the depicted time points ( a ), n = 3 mice per time point. Cytokine quantifications and statistics are relative to untreated mice (* p -value vs U). The asterisk’s color matches the cytokine legend. b – g Gray bar: PLX3397 administration window. b – g Data are Mean ± SD. Source data are provided as a Source Data file. Statistical analysis: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, the exact p-values of all comparisons are reported in the Source Data file; b One-way ANOVA vs Untreated with Dunnett post-hoc. c Two-way ANOVA with Sidak post-hoc (BU vs. BU + PLX day 40, day 90, day 210); d–g Two-way ANOVA vs Untreated with Dunnett post-hoc. Figure 2a artwork was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: ELISA assay was used to measure IL34 (DY5195-05, Mouse IL-34 DuoSet ELISA, R&D Systems) and SDF-1/CXCL12 (DY460, Mouse CXCL12/SDF-1 DuoSet ELISA, R&D Systems) cytokines in brain lysates following the manufacturer’s instructions.

Techniques: Sampling, Flow Cytometry, Luminex, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: CNS-wide repopulation by hematopoietic-derived microglia-like cells corrects progranulin deficiency in mice

doi: 10.1038/s41467-024-49908-4

Figure Lengend Snippet: a – i Analysis of 6-month-old Grn −/− (KO) mice and wild-type (WT) controls. The study scheme and timeline are depicted in Fig. . Analyses 4 months after bone marrow transplant (BMT). a GRN quantification by ELISA assay in eye lysates (WT n = 8, KO n = 5, BU + PLX n = 6). b – c GRN quantification by Western blot in eye lysates (WT n = 8, KO n = 5, BU + PLX n = 6). Tubulin was used as a loading control; the anti-GFP antibody detected the GFP protein expressed by transplant-derived cells. The uncropped blot is shown in c . d – f Analysis of proteostasis defects in KO mice. d – e Western blot analysis of ubiquitinated proteins, Cathepsin D (CTSD), and cleaved CTSD heavy chain (CTSDc/hc) in brain lysates (WT n = 6, KO n = 6, BU + PLX n = 6). Uncropped blots are shown in d and e . Tubulin was used as a loading control. f Quantification of ubiquitin and CTSD bands normalized by tubulin. g – h Targeted quantification of Bis(Monoacylglycero)Phosphate (BMP) in whole brain homogenates derived from the frontal brain. BMP levels were normalized by the total amount of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). g Heatmap showing the normalized average amounts of BMP species relative to WT mice. Absolute amounts of BMP species are reported in the Source data file. h Histogram showing the normalized amounts of total BMP species in control KO mice [KO Untreated (U), n = 5, and KO Sham (Sham) n = 6) and KO mice conditioned with BU + PLX and transplanted with WT BM (BU + PLX n = 6). Dotted diamonds indicate Sham KO mice. i Representative image of transplant-derived GFP+ cells in the frontal cortex (fCTX) of KO mice conditioned with BU + PLX. The natural GFP fluorescence is depicted. Scale bar 50 μm. The image is representative of n = 3 mice. a , b , f Data are Mean ± SD. Source data are provided as a Source Data file. Statistical analysis: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, the exact p-values of all comparisons are reported in the Source Data file; a , b , f One-way ANOVA with Tukey post-hoc; h Kruskal–Wallis with Dunn’s post-hoc.

Article Snippet: ELISA assay was used to measure IL34 (DY5195-05, Mouse IL-34 DuoSet ELISA, R&D Systems) and SDF-1/CXCL12 (DY460, Mouse CXCL12/SDF-1 DuoSet ELISA, R&D Systems) cytokines in brain lysates following the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control, Derivative Assay, Ubiquitin Proteomics, Fluorescence