EMBO/soda-nel huggingface dataset:
Astrocytic phagocytosis is a compensatory mechanism for microglial dysfunction (2020)
Figure 1

Figure 1. Clearance of microglial debris after microglial ablation.

A Time course images of the hippocampal CA1 region of Siglechdtr/dtr mice after DT administration. Sections were stained with an anti-Iba1 antibody (green) and DAPI (cyan). Arrow: live microglia with normal nucleus. Double arrow: apoptotic microglia with pyknotic nucleus. Arrowhead: microglial debris with no nucleus. Scale bar, 50 µm.

B Number of apoptotic microglia with pyknotic or fragmented nucleus (n = 5 animals per group, Kruskal-Wallis test with post hoc Dunn's test).

C Number of live microglia with normal nucleus (n = 5 animals per group, one-way ANOVA with post hoc Tukey's test).

D Number of pieces of microglial debris (Iba1+ spheres with a diameter > 2 µm and no nucleus) (n = 5 animals per group, Kruskal-Wallis test with post hoc Dunn's test).

E A representative image of a survived microglial cell in the hippocampal CA1 region of Siglechdtr/dtr mice 2 days after DT administration. Sections were stained with an anti-Iba1 antibody (green) and DAPI (cyan). A 3D image (rightmost panel) was reconstructed using Imaris software. Arrow: live microglia. Arrowhead: microglial debris. Scale bar, 10 µm.

Data information: Values show the mean ± SEM. N.S.: no significance; ***p < 0.001.
Astrocytic phagocytosis is a compensatory mechanism for microglial dysfunction (2020)
Figure 2

Figure 2. Non-microglial mononuclear populations do not act as scavengers for microglial debris.

A Histological analysis of RFP+ cells in Siglechdtr/dtr:Ccr2RFP/+ mice. Lumber spinal cord after EAE induction, and hippocampal CA1 2 or 4 days after PBS or DT injection were analyzed. Iba1 immunoreactivity (green) and RFP fluorescence (red) are shown. Images were acquired using the same laser power and sensitivity, and image processing was the same for all RFP signals (red). Scale bar, 50 µm.

B Flow cytometric analysis of RFP+Ly6Chigh inflammatory monocytes in peripheral blood of Siglechdtr/dtr:Ccr2RFP/+ mice 2 days after PBS or DT administration. Representative data and a quantification graph (n = 5 animals per group, two-tailed unpaired Student's t-test) are shown.

C Immunohistochemical localization of CD206+ perivascular macrophages (arrows) in hippocampal CA1 of Siglechdtr/dtr mice. Sections prepared 2 days after administration of PBS or DT were stained with anti-Iba1 (green), anti-CD206 (red) and anti-CD31 (blue) antibodies. Scale bar, 50 µm.

D Number of perivascular macrophages in hippocampal CA1 of Siglechdtr/dtr mice 2 days after PBS or DT administration (n = 5 animals per group, two-tailed unpaired Student's t-test).

Data information: Values show the mean ± SEM. N.S.: no significance; *p < 0.05.
Astrocytic phagocytosis is a compensatory mechanism for microglial dysfunction (2020)
Figure 3

Figure 3. Astrocyte activation after microglial ablation.

A qPCR analysis of marker molecules for CNS cell types. The hippocampus of WT and Siglechdtr/dtr mice 2 days after PBS or DT administration was analyzed (n = 3 animals per group, one-way ANOVA with post hoc Tukey's test). Results are normalized to Gapdh, and are shown as ratios to the value of WT mice injected with PBS.

B Immunohistochemical detection of astrocytes in Siglechdtr/dtr mice. Hippocampal CA1 sections were prepared 2 days after PBS or DT administration, and stained with an anti-GFAP antibody. Images were acquired using the same laser power and sensitivity, and image processing was the same. Scale bar, 30 µm.

C Astrocyte number in hippocampal CA1 of Siglechdtr/dtr mice 2 days after PBS or DT administration (n = 5 animals, two-tailed unpaired Student's t-test).

Data information: Values show the mean ± SEM. N.S.: no significance; ***p < 0.001.
Astrocytic phagocytosis is a compensatory mechanism for microglial dysfunction (2020)
Figure 4

Figure 4. Astrocytes engulf microglial debris after microglial ablation.

A Representative images of microglial debris surrounded by astrocyte processes in hippocampal CA1. Sections were prepared from Siglechdtr/dtr mice 2 days after PBS or DT administration, and stained with anti-GFAP (green) and anti-CD11b (red) antibodies. 3D images (lower row) were reconstructed from confocal images (upper row) using Imaris software. Scale bar, 10 µm.

B CLEM analysis of a phagocytic astrocyte in hippocampal CA1 of Siglechdtr/dtr mice 2 days after DT administration. A phagocytic astrocyte, which engulfed degenerated microglial components, was identified by immunohistochemistry using anti-S100β (green) and anti-CD11b (red) antibodies, and DAPI (cyan) (upper and middle panels). Then, a phagocytic cup of the same cell was analyzed by electron microscopy (lower panel). Degenerated microglial cytoplasm/plasma membrane (arrow) and nucleus (double arrow) in a phagocytic cup of the astrocyte, and a piece of microglial debris internalized in astrocytic cytoplasm (arrowhead) are indicated. Scale bar, 50 µm (upper panel), 10 µm (upper panel [inset] and middle panel), and 2 µm (lower panel).

C A representative image of a phagocytic astrocyte in hippocampal CA1 of Siglechdtr/dtr mice 2 days after DT administration. Sections were stained with anti-S100β (green) and anti-CD11b (red) antibodies, and confocal images were reconstructed using Imaris software. The lower two panels are higher magnification images of contacted microglial debris. Arrow: microglial debris contacted by astrocyte processes (coverage: approximately 30 or 50%). Arrowhead: microglial debris internalized in astrocyte cytoplasm. Scale bar, 10 µm (upper panel) and 2 µm (lower panels).

D, E Quantification of the pieces of debris (CD11b+ spheres with > 0.5 µm diameter) contacted with (coverage > 30%) (D) and internalized by (E) a single astrocyte within 30 µm-thick sections (n = 75 cells from five animals per group). The number of debris pieces was counted in 3D images reconstructed using Imaris software. All confocal images were acquired using the same laser power and sensitivity, and image processing was the same.

Data information: ***p < 0.01; Mann-Whitney U test.
Astrocytic phagocytosis is a compensatory mechanism for microglial dysfunction (2020)
Figure 6

Figure 6. Axl and Mertk in cultured astrocytes serve as phagocytic receptors for microglial debris.

A, B Cultured astrocytes engulf microglial debris. CAG-EGFP mouse-derived primary astrocytes were co-cultured with or without fluorescence-labelled apoptotic microglia. (A) Fluorescence images of GFP (green), microglial debris (red) and GFAP immunoreactivity (blue). Images were acquired using the same laser power and sensitivity, and image processing was the same in the two images (red). Scale bar, 50 µm. (B) Fluorescence images of GFP (green), microglial debris (red) and LAMP-1 immunoreactivity (cyan). Scale bar, 30 µm and 5 µm (inset).

C Flow cytometric analysis of uptake of microglial debris by cultured astrocytes. Fluorescence of internalized microglial debris was measured in GLAST+ astrocytes co-cultured with or without fluorescence-labelled apoptotic microglia.

D Expression of TAM family members and their knockdown by siRNA in cultured astrocytes. Tyro3, Axl and Metk in cultured astrocytes or hippocampal tissue were examined by western blotting. Arrows indicate the corresponding protein band(s). The secondary antibody against goat IgG generates a ~115 kDa non-specific band. The ~115 kDa band in the Axl image is the Axl-specific band overlapping the non-specific band. The ~115 kDa band in the Mertk image is the non-specific band. Axl and Mertk expression was knocked down by two siRNAs. GAPDH expression is shown as an internal control.

E Quantification of siRNA knockdown in cultured astrocytes (n = 3 independent experiments, one-way ANOVA with repeated measures). Knockdown of Tyro3 mRNA was examined by qPCR because of the very low expression level even in control astrocytes. Knockdown efficiency of Axl and Mertk was quantified from western blots. Results are normalized to Gapdh or GAPDH, and are shown as ratios to the value of non-transfected astrocytes.

F, G Flow cytometric analysis of microglial debris uptake by cultured astrocytes. After knockdown of TAM family members, astrocytes were co-cultured with fluorescence-labelled apoptotic microglia. Fluorescence of internalized microglial debris in GLAST+ astrocytes was quantified by flow cytometry (n = 4 independent experiments, one-way ANOVA with repeated measures). Results are shown as ratios to the value of non-transfected astrocytes.

Data information: Values show the mean ± SEM. N.S.: no significance; *p < 0.05, **p < 0.01; ***p < 0.001.
Astrocytic phagocytosis is a compensatory mechanism for microglial dysfunction (2020)
Figure 7

Figure 7. Astrocytes elicit their phagocytic activity upon microglial dysfunction.

A A schematic drawing of the hypothesis that astrocytes perform phagocytosis on behalf of phagocytosis-deficient microglia.

B Representative images of microglial distribution in the cerebral cortex of WT and mutant mice at 3 w. Sections were stained with anti-Iba1 or anti-CD11b antibodies (red). Scale bar, 100 µm.

C Quantification of microglial number (n = 5 animals per group, one-way ANOVA). Note that Irf8-/- mice were analyzed by CD11b immunostaining, while other strains were analyzed by Iba1 staining.

D Representative images of a spontaneous apoptotic cell and surrounding glial cells in the cerebral cortex of WT and Irf8-/- mice at 3 w. Sections were stained with anti-GFAP (green), anti-CD11b (red) and anti-cleaved caspase-3 (blue) antibodies. 3D images (lower row) were reconstructed from confocal images (upper row) using Imaris software. Arrows indicate cleaved caspase-3+ apoptotic cells. Insets are higher magnification images around apoptotic cells. Scale bar, 20 µm and 5 µm (insets).

E Percentage of spontaneous apoptotic cells surrounded by microglia in WT and mutant mice. 3D images reconstructed from confocal images were analyzed (n = 36-50 cells from five animals per group, Kruskal-Wallis test with post hoc Dunn's test).

F Percentage of spontaneous apoptotic cells surrounded by astrocyte processes in WT and mutant mice (n = 36-50 cells from five animals per group, one-way ANOVA with post hoc Tukey's test).

Data information: Values show the mean ± SEM. N.S.: no significance; ***p < 0.001.

Expanded View Figure legends
HP1gamma binding pre-mRNA intronic repeats modulates RNA splicing decisions (2021)
Figure 1

Figure 1. HP1γ associates with RNA on chromatin.

A. Scheme of the strategy used to assay HP1γ association with RNA on chromatin.

B. Genome views of RNA read density profiles on representative loci enriched (left) or low (right) in FLAG-HP1γ RNAchIP (IP) relative to input RNA (inp.) in overlaid replicate samples from HP1γ cells stimulated or not with PMA. Orange arrow, position of the locus analyzed in D.

C. Genome-wide scatter plot of IP and input normalized (norm.) RNA read counts per gene for the mean of the triplicates; n=21,754; r, Pearson's correlation coefficient between IP and input.

D. Relative quantities of RNA in the RNAchIP samples, detected by RT-qPCR with primers aligning on the intron1 of the Fosl1 gene as depicted in B (orange arrow). n=3 independent experiments.

E. Heat maps of RNA IP and input signal centered on the summit of RNAchIP peaks of IP versus input detected by MACS2 analysis (10,013 and 2609 peaks) for a representative replicate (replicate 1). Black arrows represent peak summit.

F. Percentage of RNAchIP peaks overlapping with indicated chromatin features in MEF samples from ENCODE database (green bars). Overlaps was evaluated by comparison with the list of merged peaks whose genomic location was randomized among genes with one hundred permutations (random, gray bars).

Data information: Histograms represent mean and s.d. Dots represent individual data points. P-values indicate significantly higher difference between IP in HP1γ and in KO cells (***P < 0.001; two-tailed Student's t-test).
HP1gamma binding pre-mRNA intronic repeats modulates RNA splicing decisions (2021)
Figure 2

Figure 2. HP1γ-associated RNA is enriched in CACACA and GAGAGA motifs.

A. Consensus motifs discovered among RNAchIP peaks with the RSAT pipeline, together with the percentage of peaks containing at least one exact hexameric CACACA or GAGAGA motifs or both. e-val. represents the expected number of patterns which would be returned at random for a given probability.

B. RNAchIP peaks are enriched in CACACA and GAGAGA motifs. Number of peaks containing at least one exact hexameric motif as indicated, compared to the average number of motifs in peaks whose genomic location was randomized among genes with ten permutations (random, light bars, represent mean and s.d.).

C, D. Distribution profiles of average RNAchIP signal in both IP (warm colors) and input (blue colors) over ±2kb centered on intronic (C) and exonic (D) CACACA hexameric motifs oriented in the same orientation (sense) or in opposite orientation (antisense) relative to the overlapping annotated transcript. The antisense CACACA motifs were obtained by querying the transcript sequences with the TGTGTG motif.

E. Left, representative example of a locus on the Ppp3ca gene surrounding an RNAchIP merged peak, showing the RNAchIP read density as in Fig 1B. Red and orange arrowheads correspond to oriented CACACA and GAGAGA motifs, respectively. Right, sequence of the RNA surrounding a CACACA motif highlighted in red, as well as a second a neighboring imperfect motif. The sequence was used to design a CACACA-containing RNA oligonucleotide probe (CACACA), compared to a control probe (no-CA) devoid of any related motif.

F, G, H. Gel mobility shift assays of bacterially expressed, purified HP1γ-Hinge domain, fused to GST proteins, and tested for its direct interaction with the Cy3-labeled RNA (F and H) or dsDNA (G) oligonucleotide probes depicted in (C). The probes in the gels, either free (white arrowheads) or as shifted protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.
HP1gamma binding pre-mRNA intronic repeats modulates RNA splicing decisions (2021)
Figure 5

Figure 5. Intronic RNA binding by HP1γ has an impact on RNA splicing.

A, B. HP1γ-dependent differential splicing junctions detected by MAJIQ between transcriptomes of HP1γ and KO cells. Top, Schemes of splice junctions showing annotated alternative splicing events (blue and green, panel A), or an annotated exon-exon junction and de novo cryptic junctions increased in KO (red and green, respectively, panel B).

C. Box plot depicting the length of exon junctions found differentially regulated by MAJIQ between HP1γ (green) and KO (orange) in three categories as in Fig EV5A, all classified differential HP1γ/KO junctions, annotated alternative exon junctions, non-annotated de novo cryptic junctions detected in KO only; n, differential events. Box boundaries represent 25th and 75th percentiles; center line represents median; whiskers indicate ±1.5×IQR; points are values of outliers.

D. RNAchIP read density per kb on the indicated introns for input and IP in samples from both untreated and PMA stimulated cells, n=6 biological replicates, or 3-times randomized intervals, n=18 samples. Dots represent individual data points. HP1γ/KO introns are all 272 intronic intervals between exon junctions differentially detected in HP1γ and KO. Random introns are a matching library of random intronic intervals.

E. RNAchIP read density per kb on the genes corresponding to the introns tested in (D), and analyzed as in (D), n=6 biological replicates, or 3-times randomized intervals, n=18 samples.

F. Reads from input and IP counted on the indicated features in combined unstimulated and PMA stimulated triplicate samples, n=6 biological replicates. Dots represent individual data points.

G. Average distribution profiles of sense SINE repeats on indicated exons and on ±0.7kb of surrounding introns.

Data information: All graphes represent mean and s.d. (** P < 0.005, ***P < 0.001; two-tailed (C, D, E) or one-tailed (F) Student's t-test).
HP1gamma binding pre-mRNA intronic repeats modulates RNA splicing decisions (2021)
Figure 6

Figure 6. Mutation of CACACA motifs impairs the impact of HP1γ on the splicing of a reporter minigene

A. Scheme of the minigene constructs containing the Prl exon and surrounding introns (blue) between human beta globin exons (dark grey), and containing CACACA motifs (red boxes; Prl3 wt) or mutant versions (light grey boxes; Prl3 CAmut). Orange arrowheads, position of the primers used in the assay performed in B.

B. RT-PCR analysis of the RNAs produced in the splicing assay with the Prl3 wt or Prl3 CAmut constructs as depicted in A transfected into HP1γ or KO cell lines. The top 210bp and bottom 120bp amplicons correspond to the included form (beta globin with Prl exon, more included in lane1, blue arrow, compared to lane 2), and the skipped form (beta globin exons only) of RNA, respectively. Orange arrowheads, position of the HBGEX-F and HBGEX-R primers used in PCR analysis.

C. Histogram showing the loss of inclusion of the Prl3 exon upon depletion of HP1γ (in KO) relative to inclusion in the presence of HP1γ with the Prl3 wt (red) or Prl3 CA mut (grey) reporters. Based on the quantitation of 4 experiments as the one depicted in B. Values represent the fold change relative to HP1γ as a reference (dashed line). Graph represents mean and s.d. (*** P < 0.001; two-tailed Student's t-test; n=4).

D. Model of HP1γ-dependent impact on splicing events via its binding to repeated intronic hexameric motifs (red arrows).

EXPANDED VIEW FIGURE LEGENDS
Autonomous aggregation suppression by acidic residues explains why chaperones favour basic residues (2020)
Figure 4

Figure 4: Dissection of the energetic contributions to GK efficiency.

A) Left panel: overlay of the models resulting from the introduction of Arg and Asp into the 2M5N zipper structure, with the identifier indicated for each strand. Strands E and F are highlighted in red. Right panel: the same structure turned 90° about both the x- and y-axes. Arrows in (a) indicate the capacities of Arg and Asp to distance their charged moieties. (b) shows an example of planar Arg stacking.

B) Boxplots showing all-atom analysis of the energetic effects of the introduction of GKs into a set of amyloid steric zipper structures. The upper whiskers indicate the largest value no further than 1.5 times the Inter-Quartile Range (IQR) from the upper hinge, the lower whiskers show the smallest value at least 1.5 * IQR from the lower hinge. Notches extend 1.58 * IQR / sqrt(n) from either side of the median and represent a 95% confidence interval for the median value. Grey shaded area indicates total energy associated with the introduction of GKs into zipper structures, the remainder of the plot shows the breakdown of this total energy in into constituent energy terms. Each boxplot shows the distribution of 72 observations.

C) Violin plots showing the distribution of distances to the closest charged moiety in a neighboring GK residue in the central-most positions (strands E and F) of the steric zipper models. Each violin shows the distribution of 83 observations.

D) Time-resolved Dynamic Light Scattering (DLS) analysis showing average particle radius over time for the β-Gal peptides flanked by both natural and non-natural GKs, at increasing NaCl concentrations (indicated in µM). Structures of the amino acids flanking the aggregating core are indicated on the right-hand side, the sequence of the aggregating core is indicated below.
Autonomous aggregation suppression by acidic residues explains why chaperones favour basic residues (2020)
Figure 6

Figure 6: DnaK compensates for the inferior gatekeeping potential of basic residues.

A) DnaK binding to cellulose-bound peptide array. Sequences of the aggregating cores are indicated in the top of the panel, the GKs flanking them in each instance are indicated above the blots.

B) Schematic representation of folding-incompetent model protein. The β-Gal APR is flanked on either side by one of the charged GKs, and separated from the GFP moiety by a rigid linker. Additionally, the construct is C-terminally His-tagged.

C) Representative Western blot of in vitro translation analysis of folding-incompetent model constructs. Bands show Total (T) and Soluble (S) fractions after centrifugation at 21,000 g for 30 mins. "+ Dnak" indicates addition of either DnaK mix - containing DnaK, DnaJ and GrpE.

D) Bar plots showing mean solubility as determined through quantification of western blot in (C), whiskers indicate standard deviation (n=4). Statistically significant differences between the -DnaK and +DnaK conditions were determined through two-way ANOVA with Bonferroni's correction for multiple comparisons. P-values for significant differences are indicated.

E) Representative western blot of the solubility analysis of GK variants upon overexpression in E. coli K12. Bands show Soluble (Sol) and Pellet fractions after centrifugation at 17,100 g for 15 mins. "-DnaK" indicates expression in E. coli K12 DnaK knockout line, "+ Dnak" indicates co-expression of pKJE7, encoding DnaK, DnaJ and GrpE.

F) Barplots showing mean solubility as determined through quantification of western blot bands in (E), whiskers indicate standard deviation (n=7, except for the AA construct, where n=4). Statistical significance was determined through two-way ANOVA followed by Tukey's multiple comparisons test. P-values are indicated.

G) Structured Illumination Microsopy (SIM) images of E. coli after three hours of expression of one of the GK variants. GFP fluorescence is shown in green. "ΔDnaK" indicates expression in E. coli K12 DnaK knockout line, "+ Dnak" indicates co-expression of pKJE7, encoding DnaK, DnaJ and GrpE.