Journal: bioRxiv

Article Title: Mechanism of neurodegeneration mediated by clonal inflammatory microglia

doi: 10.1101/2024.07.30.605867

Figure Lengend Snippet: (A) Schematic of the brains of the 8 patients annotated for the detection of BRAFc.1799T>A (V600E) and/or of histological signs of neurodegeneration (Histo+). Bar graphs represents the proportion of tested brain samples positive for BRAFc.1799T>A (V600E) by HemePACT and/or histological signs of neurodegeneration among patients with (left) or without (right) neurological symptoms . (B) . Left, representative H&E, IBA1 (microglia marker) and GFAP (astrocyte marker) of pons and cerebellum from patient #1 and an age-match control for comparison. Right, representative IBA1 and GFAP of pons from patient #8, #4, #5, #7 and an age-match control for comparison. Arrows indicate neuron nuclei. (C) Pathway enrichment among differentially expressed genes (DEG, red: upregulated genes, blue: downregulated genes) by RNAseq analysis (FDR <0.05, log2FC >= or <= 1.5/-1.5) of whole brain of Histiocytosis patients (n=13) and controls (n=11) using g:profiler webtool. Pathways are selected based on FDR <= 0.05 and ordered by significance. (D) Hierarchical clustering of DEG (log2FC >= 1.5, log2FC <= −1.5, FDR <0.05) between brain samples from Histiocytoses (n=13) and controls (n=11). Expression values are Z score transformed. (E) Bar graphs represents the proportion of tested brain samples positive for BRAFc.1799T>A (V600E) by HemePACT and/or histological signs of neurodegeneration among patients with (left) or without (right) neurological symptoms. (F) Variant allelic frequency (VAF, %, HemePACT)) for BRAFc.1799T>A (V600E) in PU.1+, samples from patients with (red) and without (blue) neurological symptoms. Each symbol represents a patient. Statistics: p-value is calculated using a mixed-effects linear regression model (see methods).

Article Snippet: Immunofluorescence of mouse tissue was carried out on 3–4-μm thick paraffin sections, fixed with PFA with anti-pSTAT3-Tyr705 (1:100 D3A7, XP® Rabbit mAb, Cell Signaling), anti-IBA1 (1:200, AIF-1/IBA1 Antibody Goat Polyclonal antibody, Novus Biologicals) and anti-GFAP (1:200, ab4674, Chicken polyclonal, Abcam), NEUN (1:200, MAB377, Millipore).

Techniques: Marker, Control, Comparison, Expressing, Transformation Assay, Variant Assay

Journal: bioRxiv

Article Title: Mechanism of neurodegeneration mediated by clonal inflammatory microglia

doi: 10.1101/2024.07.30.605867

Figure Lengend Snippet: (A) H&E, IBA1 (microglia marker) and GFAP (astrocyte marker) staining of hippocampus and frontal cortex from patient #1 and an age-matched control individual for comparison. (B) For patient #1, PET scan (top) at age 9 shows hypometabolism in the cerebellum (arrow) and the thalamic region MRI at age 18 (bottom) shows hyperintensity signals in pons, dentate nuclei, and the cerebellum (arrows). MRI images show hyperintensity signals in the cerebellum dentate nuclei and cerebellar pedunculus of patient 2,3,8,4 and 5 (arrows). Patient 7 MRI did not reveal detectable abnormalities. No MRI was available for patient 6. (C) Bar graphs represent the proportion of tested brain samples positive for BRAFc.1799T>A (V600E) by droplet-digital PCR (ddPCR) and/or histological signs of neurodegeneration among patients with (left) or without (right) neuro-histiocytosis. (D) Representative H&E from pons and cerebellum from patients ranked by neuronal damage by two pathologists, from higher to lower.

Article Snippet: Immunofluorescence of mouse tissue was carried out on 3–4-μm thick paraffin sections, fixed with PFA with anti-pSTAT3-Tyr705 (1:100 D3A7, XP® Rabbit mAb, Cell Signaling), anti-IBA1 (1:200, AIF-1/IBA1 Antibody Goat Polyclonal antibody, Novus Biologicals) and anti-GFAP (1:200, ab4674, Chicken polyclonal, Abcam), NEUN (1:200, MAB377, Millipore).

Techniques: Marker, Staining, Control, Comparison, Digital PCR

Journal: bioRxiv

Article Title: Mechanism of neurodegeneration mediated by clonal inflammatory microglia

doi: 10.1101/2024.07.30.605867

Figure Lengend Snippet: (A) Variant allelic frequency (VAF, %, by HemePACT) of BRAFc.1799T>A (V600E) in PU.1+ nuclei from histiocytosis patients across brain regions (n=8, patients with neuro-histiocytosis are color-coded in red, patients without a diagnosis of neuro-histiocytosis are color-coded in blue). The fitted line, R-squared and corresponding p value were calculated by simple linear regression by assigning numbers from 1-8 to each brain region from along a rostro caudal axis. Gray line: all patients. Red line: patients with neuro-histiocytosis. Blue line: patients without neuro-histiocytosis. (B) Representative mouse sagittal midline brain sections from 6 months old Csf1r MerCreMer ; Braf LSL-V600E mice (pulsed with OH-TAM at E8.5 ), Cx3cr1 CreERt2 ; Braf LSL-V600E (pulsed with OH-TAM at E9.5) and littermate controls stained with anti-IBA1 or anti-GFAP, Scale bar 1000uM. (C) Allelic frequency of the Braf V600E allele in microglia purified from dissected brain regions from 2 months old, and at 6-12 months-old analyzed by droplet digital PCR (ddPCR). Dots and colored lines represent individual mice, boxes represent variance, with line at mean. (D) RNAseq analysis performed in FACS-isolated microglia from cortex and brainstem from 2-month-old Cx3cr1 CreERT2 Braf LSL-V600E mice (n=3), and littermates (n=3) pulsed with OH-TAM at E8.5. Top, principal component analysis (PCA). Bottom, pathway analysis of significantly upregulated genes (FDR <0.05, log2FC >= 1.5) in microglia from old Cx3cr1 CreERT2 Braf LSL-V600E versus littermate control using g:profiler webtool. Pathways are selected based on FDR <= 0.05 and ordered by significance. (E) Hierarchical clustering of DEG from ‘mitotic cell cycle process’ (GO:1903047, left) from analysis in D.

Article Snippet: Immunofluorescence of mouse tissue was carried out on 3–4-μm thick paraffin sections, fixed with PFA with anti-pSTAT3-Tyr705 (1:100 D3A7, XP® Rabbit mAb, Cell Signaling), anti-IBA1 (1:200, AIF-1/IBA1 Antibody Goat Polyclonal antibody, Novus Biologicals) and anti-GFAP (1:200, ab4674, Chicken polyclonal, Abcam), NEUN (1:200, MAB377, Millipore).

Techniques: Variant Assay, Staining, Purification, Digital PCR, Isolation, Control

Journal: bioRxiv

Article Title: Mechanism of neurodegeneration mediated by clonal inflammatory microglia

doi: 10.1101/2024.07.30.605867

Figure Lengend Snippet: (A) Variant allelic frequency (VAF, %, by ddPCR) of BRAFc.1799T>A (V600E) in PU.1+ nuclei from histiocytosis patients across brain regions (n=8, patients with neuro-histiocytosis are color-coded in red, patients without a diagnosis of neuro-histiocytosis are color-coded in blue). The fitted line, R-squared and corresponding p value were calculated by simple linear regression by assigning numbers from 1-8 to each brain region from along a rostro caudal axis. (B) Mouse models of neuro-histiocytosis. Csf1r MerCreMer and Cx3cr1 CreERt2 pregnant females crossed with Braf LSL-V600E males receive low-dose 4-Hydroxytamoxifen (a single injection of 37.5 mg 4-OHT per kg, ip, and 18.75 mg per kg of body weight of progesterone) at E8.5 and E9.5 respectively. (C) Plots represent disease score progression and survival over time of mice in B. (D) Quantification of % of IBA1+ area from Csf1r MerCreMer Braf LSL-V600E and Cx3cr1 CreERt2 Braf LSL-V600E mice and littermate controls. Statistics: p values were calculated with unpaired t-test with Welch’s correction.

Article Snippet: Immunofluorescence of mouse tissue was carried out on 3–4-μm thick paraffin sections, fixed with PFA with anti-pSTAT3-Tyr705 (1:100 D3A7, XP® Rabbit mAb, Cell Signaling), anti-IBA1 (1:200, AIF-1/IBA1 Antibody Goat Polyclonal antibody, Novus Biologicals) and anti-GFAP (1:200, ab4674, Chicken polyclonal, Abcam), NEUN (1:200, MAB377, Millipore).

Techniques: Variant Assay, Injection

Journal: bioRxiv

Article Title: Mechanism of neurodegeneration mediated by clonal inflammatory microglia

doi: 10.1101/2024.07.30.605867

Figure Lengend Snippet: (A) Top, pathway analysis using g:profiler webtool of common differential expressed genes between 2 month old Cx3cr1 CreERt2 Braf LSL-V600E mice microglia in and human whole brain samples in . Bottom, hierarchical clustering of common genes in end stage mouse microglia. Expression values are Z score transformed. Samples were clustered using average linkage and cluster similarity was determined using the Euclidean distance . (B) Single-nuclei RNAseq (snRNAseq) analysis of dissected cortex and brainstem from Braf VE/WT Cx3cr1 CreER mice pulsed with OH-TAM at E9.5 and analyzed at 6 month of age (end stage, VE) (n=2) and littermate controls (Ctrl, n=2). After QC and data processing nuclei were clustered and annotated using Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) by cell-type. (C) Bar plot showing the relative frequency of neurons, microglia and stromal cells by brain region and condition. (D) UMAP of all nuclei color-coded by brain region (left) or by condition (right). (E) Top, UMAP of neuronal nuclei. color-coded by brain region (left) or by condition (right). Bottom, schematic of brainstem depicting the localization of neuronal clusters reduced in VE samples and NeuN staining (iDISCO) of the pons tegmental nucleus (TRN) from mutants and littermate control. Plot shows the quantification of NeuN staining by immunofluorescence in BRAFV600E and control mice. Each dot represents the mean of three fields per mouse. Statistics: p-values are calculated with Student t test. (F) Left, Dot plot showing the expression level (color scale) and the percent of cells expressing (dot size) the most significantly upregulated DEG (log2FC >= 0.5 & FDR <= 0.05) between brainstem astrocytes, A3 (VE) vs A1,2 (control). Barplot represents number of cells per cluster. Right, pathway analysis of DEG in astrocytes using enrichR of upregulated (red) and downregulated (blue) genes in cluster A3 (FDR <0.05, log2FC >= or <= 0.5/-0.5) in comparison to clusters 1,2. Plot shows the quantification of the % of pSTAT3 expressing cells by immunofluorescence, among IBA1+, GFAP+, and IBA1+ GFAP+ cells in the brainstem of Cx3cr1 CreERT2 ; Braf LSL-V600E and control mice. (G) Violin plots showing expression scores for previously defined disease-associated astrocytes signatures, across astrocytes clusters. ( H) Left, Dot plot showing the expression level (color scale) and the percent of cells expressing (dot size) the most significantly upregulated genes (log2FC >= 0.5 & FDR <= 0.05) between brainstem oligodendrocytes O2,4,5 (VE) vs O 0,1 (control). Right, pathway analysis of DEG in oligodendrocytes using enrichR of upregulated (red) and downregulated (blue) genes in cluster O2,4,5 (FDR <0.05, log2FC >= or <= 0.5/-0.5) in comparison to clusters 0,1.

Article Snippet: Immunofluorescence of mouse tissue was carried out on 3–4-μm thick paraffin sections, fixed with PFA with anti-pSTAT3-Tyr705 (1:100 D3A7, XP® Rabbit mAb, Cell Signaling), anti-IBA1 (1:200, AIF-1/IBA1 Antibody Goat Polyclonal antibody, Novus Biologicals) and anti-GFAP (1:200, ab4674, Chicken polyclonal, Abcam), NEUN (1:200, MAB377, Millipore).

Techniques: Expressing, Transformation Assay, Staining, Control, Immunofluorescence, Comparison

Journal: bioRxiv

Article Title: Mechanism of neurodegeneration mediated by clonal inflammatory microglia

doi: 10.1101/2024.07.30.605867

Figure Lengend Snippet: (A) Percentage of IBA1+ area in brain from Csf1r MerCreMer ; Braf LSL-V600E mice and littermate controls pulsed with OH-TAM at E8.5 and treated from 3 months of age with food formulated with CSF1R inhibitor (PLX-5622) n=6, Braf-V600E inhibitor (PLX-4720) n=6, both n=6, or control diet n=5. p-values were calculated with one-way ANOVA, using Dunnett’s multiple comparisons test. (B) Disease score progression and (C) survival curves for mice in A. Ticks indicate animal death/experimental endpoint. Statistics: Mantel-Cox test. P values are for comparison with control diet. Hazard ratio (logrank): for (a) [CSF1R inh/Ctrl Diet (HR 0.38; 95% CI, 0.19-0.76)], [Braf inh/Ctrl Diet (HR 0.30; 95% CI, 0.15-0.63)], [Both inh/Ctrl Diet (HR 0.20; 95% CI, 0.09 to 0.45)], for (b) [CSF1R inh/Ctrl Diet (HR 0.28; 95% CI, 0.06 to 1.24)], [Braf inh/Ctrl Diet (HR 0.18; 95% CI, 0.03 to 0.92)], [Both inh/Ctrl Diet (HR 0.16; 95% CI, 0.03 to 0.81. (D) Quantification of NeuN staining by immunofluorescence in the pons tegmental reticular nuclei (TRN) from control and Csf1r MerCreMer ; Braf LSL-V600E mice treated with control diet, BRAF inhibitor (PLX4720) CSF1R inhibitor (PLX5622) or the combination. Each dot represents the mean of three fields per mouse. p-values were calculated with ANOVA.

Article Snippet: Immunofluorescence of mouse tissue was carried out on 3–4-μm thick paraffin sections, fixed with PFA with anti-pSTAT3-Tyr705 (1:100 D3A7, XP® Rabbit mAb, Cell Signaling), anti-IBA1 (1:200, AIF-1/IBA1 Antibody Goat Polyclonal antibody, Novus Biologicals) and anti-GFAP (1:200, ab4674, Chicken polyclonal, Abcam), NEUN (1:200, MAB377, Millipore).

Techniques: Control, Comparison, Staining, Immunofluorescence

Journal: PLoS ONE

Article Title: Role of the Mitochondria in Immune-Mediated Apoptotic Death of the Human Pancreatic β Cell Line βLox5

doi: 10.1371/journal.pone.0020617

Figure Lengend Snippet: βLox5 cells were treated with the combination of rhTNFα (2000 U/mL) and rhIFNγ (1000 U/mL) for 48 h with and without pan-caspase inhibition with Z-VAD-FMK (50 µM x 2). Immunofluorescence analysis shows increased AIF translocation to the nucleus in cytokine treated cells. Representative images are shown. White arrows indicate cells with high nuclear AIF staining. *denotes statistical significance with a P value <0.05. NS denotes no statistical difference.

Article Snippet: Cells were fixed with 2% paraformaldehyde (PFA) for 10 minutes at room temperature (RT), permeabilized with 100% ice-cold methanol for 10 minutes, then blocked with 10% Normal Goat Serum (NGS) for 40 minutes at RT with single PBS washes between each step and two washes before adding the antibody. βLox5 cells were conjugated with AIF antibody (R&D Systems) for 1 hour at 37°C, washed 3 times, then stained with FITC conjugated anti-rabbit IgG (1∶200) for 60 minutes in the dark.

Techniques: Inhibition, Immunofluorescence, Translocation Assay, Staining