Additional file 1 of Cytokine enrichment in deep cerebellar nuclei is contributed by multiple glial populations and linked to reduced amyloid plaque pathology
Creators: name: Gaunt, Jessica R.
name: Zainolabidin, Norliyana
name: Yip, Alaric K. K.
name: Tan, Jia Min
name: Low, Aloysius Y. T.
name: Chen, Albert I.
name: Ch’ng, Toh Hean
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. Schematic of EC and DCN dissection. Figure S2. Workflow for snRNAseq. Figure S3. SnRNAseq breakdown of number of cells per cell type and condition. Figure S4. Proportion of cells of each type isolated for each condition. Figure S5. Pseudo-bulk heatmaps showing average expression of genes differentially expressed between APP and WT genotypes in the EC or DCN for each cell type. Heatmaps show expression in each condition in all major cell-types with z-scores calculated for each gene (row). Conditions and cell-types are indicated by colour-coded bar above each heatmap. DEGs in the EC and DCN respectively are indicated by colour-coded bars to the left of each heatmap. Hierarchical clustering was applied to genes in each heatmap. (A) Astrocyte (B) Neurons (C) Microglia (D) Oligodendrocytes (E) OPCs (F) Vascular cells. Figure S6. (A) UMAP of module Mg.G4 as a percentage of the maximum expression of genes found in the module. (B) Expression UMAPs for selected top marker genes from module Mg.G4 showing percentage of maximum expression. (C) Gene Ontology analysis of biological processes enriched in module Mg.G4 compared to all genes detected in microglia. Figure S7. (A) Expression pattern of Hexb (left panels) in the cortex and the cerebellum. On the right is an illustration of Hexb aggregates sized between 10 to 100 µm2 marked by grey dots while aggregates sized larger than 100 µm2 marked in red. Dashed lines in the cortical region indicates the EC region. Dashed lines in the cerebellum demarcates the DCN region while the grey shaded area denotes the white matter (WM) region. Scale bar indicates 1000 µm. (B) Quantification of the number of Hexb population in the EC, DCN and WM of WT and APP tissue (N=3 subjects, n=6-7 sections/subject). Graph indicates mean ± SEM. (C) RNA in situ hybridisation of Oasl2 and Ifi204 (green) in the DCN and WM of APP tissue. Closed circle denotes colocalisation with Hexb (red) while broken circle denotes absence of Oasl2 or Ifi204 in Hexb-positive cells. Scale bar represents 10 µm. (D-E) Quantification of Oasl2-expressing microglia (D) and Ifi204-expressing microglia (E) in the DCN and WM (N=3 subjects, n=3 sections/subject). Graphs indicate mean ± SEM. Student’s t-tests were performed on within region (*) and within-genotype differences in DCN or WM as compared to EC (†). Two-way ANOVAs were performed on within-subject means to test for main effects of region and genotype and interaction effects, followed by Tukey’s HSD post hoc tests (¤). * p&lt;0.05, ** p&lt;0.01, *** p&lt;0.001, **** p&lt;0.0001. Figure S8. (A-D) UMAPs showing (i) cell subclusters, (ii) distribution of cells across conditions and (iii) aggregated expression of genes in each module, and (iv) box plots of log2 gene expression value distributions for each gene module for (A) Astrocytes, (B) Oligodendrocytes, (C) OPCs and (D) Vascular cells. Figure S9. Dot plots showing expression of select DEGs associated with innate immune responses, cytokine production and NFKB signalling in modules Vasc.2, Vasc.4, and Olig.3 across conditions. Dot size indicates the percentage of cells in each condition expressing each gene and dot colour indicates mean log2 expression. Darker dots represent higher expression levels. Figure S10. Cytokine protein expression measured by multiplex array in WT and App KI tissues. Bar plots show mean and SEM for each condition with dots for individual sample measurements. Region and genotype are indicated by bar colour and shade. Significant post hoc t-test results are indicated as follows: * p&lt;0.05, ** p&lt;0.01, *** p&lt;0.001, **** p&lt;0.0001. (A) EC &amp; DCN. (B) Cortex (CTX) and cerebellum (CBM). Figure S11. STRING analysis of DEGs that overlap in DCN and LPS conditions compared to controls in BV2-treated cells. Genes associated with GO label “immune response”, “response to cytokines” and “inflammatory response” are represented as colour coded circles. (Gene interactions shown under medium confidence with no disconnected nodes.). Figure S12. Log2 fold change in expression of marker genes associated with inflammation in BV2 microglia cultures stimulated with DCN homogenates compared to EC homogenates from the same subject, measured by qPCR (N=8). P-values from paired t-tests are written above each bar. Figure S13. Cytokine protein expression measured by multiplex array following microglia depletion by CSF1R inhibition. WT and App KI mice were fed with PLX5622 or control chow for 2 months, followed by isolation of the EC and DCN. Bar plots show mean and SEM for each condition with dots for individual sample measurements. Region and genotype are indicated by bar colour, while treatment is indicated by diagonal stripes. Protein concentrations are normalised by total protein in each sample. Figure S14. Quantification of IBA1+ microglia coverage in the EC and DCN of WT mice fed with PLX5622 and control chow, showing robust depletion in both regions (N=4 subjects, n=2 sections/subject). * p&lt;0.05, *** p&lt;0.001. Figure S15. Quantification of 6E10-labeled amyloid plaques in the EC (A) and DCN (B) for the number, total area and average size of two different plaque size thresholds (&gt;10 µm2 and &gt;100 µm2; N=9 subjects, n=3 sections/subject). Figure S16. Expression of App and select APP-processing genes in snRNAseq data. (A) UMAPs displaying log2 expression of App across all cells in each condition. (B) Violin plots of App expression in each major cell type and condition. (C) Heatmap of pseudo-bulk average gene expression of App and select APP-processing genes in each major cell type in each condition. Colour scale indicates z-scores for each gene across conditions.