Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: (A) Genetic strategy targeting exon 4 of the Chd8 gene. The exon was flanked with loxP sites to excise the loci, resulting in a frameshift mutation that disrupts the production of the CHD8 protein after Cre excision. (B and C) Representative images of CHD8 in the CA1 regions of adult mouse brains. CHD8 is expressed in neurons and astrocytes in controls (B). After tamoxifen administration, Chd8 fx/fx : CAGGS-CreER +/ − mouse brains showed non-detectable levels of CHD8 protein (C). In both (B) and (C), arrows point to astrocytes expressing CHD8 and its knockout thereafter. (D) Schematic diagram for tamoxifen-induced Chd8 cKO, the stab-wound injury model, and the analysis of reactive gliosis. (E and F) Response of GFAP + astrocytes and Iba1 + microglia after stab-wound injury. Control mice (E) exhibit astrocytic and microglial response as expected, while global Chd8 cKO mice (F) show reduced staining for both GFAP + and Iba1 + along the needle track. (G and H) High-magnification images from the injury site from the corresponding genotypes. Note the reduction in cell body size, process elongation, and polarity in astrocytes from global Chd8 cKO mice (H). (I and J) Decreased area occupied by GFAP + astrocytes (I) and Iba1 + microglia (J) in global Chd8 cKO mice compared to controls. In (B) and (C), scale bars indicate 20 μm; in (G) and (H), scale bars indicate 50 μm; in (E) and (F), scale bars indicate 500 μm. The dashed rectangle indicates the ROIs that were quantified. In (E)–(H), the dashed lines indicate the needle track of the injury. In (I) and (J), data points illustrate the quantified area from the six brain slices most proximal to the injury epicenter. Data are normalized to the means of the ipsilateral site in control mice. **** p < 0.0001, ns, not significant; statistical analysis was performed with two-way ANOVA; on the violin plots, dashed lines indicate the 25%, mean, and 75% percentile, respectively, from bottom to top; n = 4 mice per genotype.

Article Snippet: CHD8 Rabbit polyclonal 1 to 1000 , Novus Biologicals , NB 100-60418.

Techniques: Mutagenesis, Expressing, Knock-Out, Control, Staining

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: (A) Strategy for tamoxifen-induced, astrocyte-specific Chd8 cKO utilizing the Aldh1l1-CreERT2 line. Mice were crossed with the Ai14- tdTomato reporter line to visualize recombined cells. (B and C) Astrocyte-specific Chd8- cKO mice show non-detectable CHD8 protein expression in cortical slices. Controls are mice without the Chd8 floxed alleles but expressing Aldh1l1-CreERT2 to turn on the expression of tdTomato reporter. Arrows indicate CHD8 expression in tdTomato + astrocytes in control but not astrocyte Chd8 cKO. (D and E) GFAP and Iba1 staining of astrocytes and microglia, respectively, after stab-wound injury in control (D) and astrocyte cKO mice (E). (F and G) High-magnification images from the injury sites from the corresponding genotypes showing reduction in cell body size, process elongation, and polarity in astrocytes and reduced microglia numbers in astrocyte cKO mice. (H and I) Decreased area occupied by GFAP + astrocytes and their numbers in astrocyte cKO mice compared to controls. (J and K) Decreased area occupied by Iba1 + microglia and their numbers in astrocyte cKO mice compared to controls. In (H)–(K), data points represent the quantified area from the six brain slices most proximal to the injury epicenter. Data are normalized to the means of the ipsilateral site in control mice. In (B) and (C), scale bars indicate 20 μm; in (F) and (G), scale bars indicate 50 μm; in (D) and (E), scale bars indicate 500 μm. The dashed rectangle indicates the ROIs that were quantified. In (D)–(G), the dashed lines in merged images indicate the needle track of the injury. In (H)–(K), *** p < 0.001; **** p < 0.0001; ns, not significant; statistical comparisons were analyzed with two-way ANOVA; on the violin plots, dashed lines indicate the 25%, mean, and 75% percentile, respectively, from bottom to top; n = 6 mice per genotype.

Article Snippet: CHD8 Rabbit polyclonal 1 to 1000 , Novus Biologicals , NB 100-60418.

Techniques: Expressing, Control, Staining

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: (A) Heatmap of DEGs identified through RNA-seq between control and astrocyte Chd8 cKO mice after LPS treatment ( n = 5 mice per genotype). A total of 109 DEGs were identified, 76 of which were upregulated and 33 were downregulated (FDR < 0.05). (B) Volcano plot depicting the distribution of upregulated and downregulated genes, relative to their quantified fold change and their corresponding p values. The threshold was set at p (adjusted) < 0.05. (C) Venn diagram of the detected DEGs, depicting a subset of DEGs that correspond to genes whose expression is specific to astrocytes, neurons, and microglia. Of those, many DEGs (41) were determined to be astrocyte specific, while fewer were deemed to be specific in neurons (4) and microglia (1). (D) Bar plot showing the fold enrichment of the detected DEGs in our dataset, indicating significant enrichment of DEGs for astrocyte marker genes. Notably, no enrichment was detected when comparing neuronal or microglial genes to the cell-type markers from previous studies, as cited. *** p < 0.001; **** p < 0.0001. (E) GO terms analysis reveals changes associated with many cellular processes, including lipid and metabolic pathways in astrocyte cKO mice in response to LPS stimulation. (F) Heatmap of the top 20 DEGs identified through RNA-seq. Of these, 13 were upregulated and 7 were downregulated in astrocyte cKO mice treated with LPS vs. control mice treated with LPS. (G–O) qPCR analysis of Gstt3 (G), Acsl3 (H), Etnppl (I), Phykpl (J), Gjb6 (K), Slc9a8 (L), Agt (M), Tnfrsf25 (N), and Lcat (O) mRNA confirms the altered expression shown in (F) ( n = 4 mice per group). (P) Representative western blotting of TNFRSF25 from cortices of control and astrocyte cKO mice after LPS administration, showing reduced TNFRSF25 protein in the cortex of astrocyte cKO mice ( n = 7 mice per group). (Q) Representative western blotting of LCAT from cortices of control and astrocyte cKO mice after LPS administration showing reduced LCAT protein in astrocyte cKO mice ( n = 7). (R) Representative images of TNFRSF25 staining in the cortex of control and astrocyte cKO mice after LPS administration. Signal intensity quantification of TNFRSF25 staining ( n = 4 mice per genotype). * p < 0.05; scale bars indicate 200 μm. Data points are normalized to the mean of the control group. Error bars depict the SEM. Statistical comparisons were performed with the one-tailed Welch’s t test.

Article Snippet: CHD8 Rabbit polyclonal 1 to 1000 , Novus Biologicals , NB 100-60418.

Techniques: RNA Sequencing, Control, Expressing, Marker, Western Blot, Staining, One-tailed Test

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: (A) Schematic diagram illustrating the elements required for the designed AAV for astrocyte-specific Chd8 editing in vivo via CRISPR-SaCas9. (B) Diagram for the simultaneous AAV injection and stab-wound injury with analysis of reactive gliosis performed at 7 days post-injection. (C) Representative images near the needle track from control mice (Scramble-AAV injected). CHD8 is detectable in astrocytes (SOX9 + ) expressing SaCas9 (HA tag + ). (D) Representative images near the needle track from mice injected with the Chd8 -cKO AAVs. CHD8 is undetectable in the majority of HA + and SOX9 + astrocytes (white arrows), while fewer HA + and SOX9 + astrocytes still show CHD8 expression (yellow arrows). (E and F) GFAP and Iba1 staining of astrocytes and microglia, respectively, after stab-wound injury and AAV injection in the Scramble-AAV (E) and Chd8 -cKO-AAV groups (F). (G) Decreased area occupied by GFAP + astrocytes in the Chd8 -cKO-AAV mice. (H) Quantification of the area occupied by Iba1 + microglia between control and Chd8 -cKO-AAV mice. In (C) and (D), scale bars indicate 20 μm; in (E) and (F), scale bars indicate 500 μm. The dashed rectangle indicates the ROIs that were quantified in (G) and (H). The dashed lines in merged images indicate the needle track of the injury. In (G) and (H), statistical comparisons were performed with two-way ANOVA; ** p < 0.01. Data points indicate n = 3 mice per group. Data are normalized to the means of the ipsilateral site in the control group.

Article Snippet: CHD8 Rabbit polyclonal 1 to 1000 , Novus Biologicals , NB 100-60418.

Techniques: In Vivo, CRISPR, Injection, Control, Expressing, Staining