Article Figures & Data
Figures
- Figure 1.
Single-nucleus quality metrics do not differ between fresh 0 and 3 h postmortem interval (PMI) mice. A, Schematic representation of the experimental setup. Single-nucleus RNA libraries were generated from the brains of 37-week-old C57BL/6J wild-type (N = 9; WT) and tau P301S (N = 9; PS19) mice. Samples were collected immediately post-killing (N = 8; 0 h PMI) or 3 h postmortem (N = 10; 3 h PMI). B, Bar graph showing the total nuclei count per group, categorized by PMI and genotype. C, The percentage graph illustrating the distribution of nuclei across different cell cycle phases (G1, S, G2/M) is similar across all groups. D, UMAP visualizing the clustering of annotated cell types. Sample information and RNA integrity number (RINe) in Extended Data Table 1-1. Verification of the expression of human MAPT in PS19 mice and the genetic sex of the mice were investigated, as shown in Extended Data Figure 1-1. Group and individual quality control metrics are shown in Extended Data Figure 1-2. Extended Data Figure 1-3 further shows cell type abundance among samples. Extended Data Figure 1-4 displays the expression of cell type markers within each identified cell type. Extended Data Figure 1-5 displays the principal component analysis (PCA) of the aggregated expression values at the sample and cell type level, which shows a clear delineation of the major cell types.
- Figure 2.
Genes differentially expressed between 3 h PMI and 0 h fresh samples within the neuron cell types are involved in immune response and stress pathways. A, Volcano plot of differentially expressed genes (DEGs) in neuron cell type, comparing 3 h PMI, N = 10 versus fresh 0 h, N = 8 samples (total N = 18). Downregulated genes (log2 fold change < 0, q < 0.1) are shown in green, upregulated genes (log2 fold change > 0, q < 0.1) in purple, and nonsignificant genes (q ≥ 0.1) in gray. B, Gene ontology (GO) analysis showed an upregulation of cellular response, circadian rhythm, inflammatory response, and positive regulation of the apoptotic process. Downregulated genes are enriched in regulating metabolic processes, cell secretion, and influenza infection. The x-axis is the GO term, and the y-axis is the −log10 p value. C, D, Similarly, genes upregulated within the interneuron cell type between 3 h PMI and fresh 0 h are enriched in response to oxidative stress and regulation of synaptic plasticity.
- Figure 3.
Genes differentially expressed between 3 h PMI and 0 h fresh samples within the glial cell types are involved in rhythmic processes and cell–cell adhesion pathways. A, Volcano plot of differentially expressed genes (DEGs) in astrocyte cell type, comparing 3 h PMI, N = 10 versus fresh 0 h, N = 8 samples (total N = 18). Downregulated genes (log2 fold change < 0, q < 0.1) are shown in green, upregulated genes (log2 fold change > 0, q < 0.1) in purple, and nonsignificant genes (q ≥ 0.1) in gray. B, Gene ontology (GO) analysis shows the downregulation of behavior and rhythmic processes. The x-axis is the gene count contributing to the enrichment pathways listed on the y-axis. The color of the bar indicates the −log10 p value. C–F, Similarly, genes downregulated or upregulated within the other glial cell types, including oligodendrocytes, fibroblast, and OPC are enriched in cell–cell adhesions, GTPase cycle, brain development, and synaptic signaling. Extended Data Figure 3-1 displays the transcriptomic effect of PMI 3 versus 0 h PMI for endothelial, mural, and microglia cell types.
- Figure 4.
Reduced PS19 versus WT differential expression signal at the 3 h PMI compared with 0 h fresh samples. A, Volcano plot of differentially expressed genes (DEGs) in neuron cell type, comparing PS19 (N = 4) versus WT (N = 4) in fresh 0 h samples (total N = 8). Downregulated genes (log2 fold change < 0, q < 0.1) are shown in blue, upregulated genes (log2 fold change > 0, q < 0.1) in red, and nonsignificant genes (q ≥ 0.1) in gray. B, Volcano plot of DEGs in neuron cell types, comparing PS19 (N = 5) versus WT (N = 5) in 3 h PMI samples (total N = 10). C, UpSet plot depicting shared and unique DEGs between 0 and 3 h PMI analyses. Panels D–F present the corresponding analyses for interneurons and panels G–I for astrocytes. Extended Data Figure 4-1 displays the gene ontology (GO) enrichment analysis of upregulated and downregulated genes in PS19 versus WT at 0 and 3 h postmortem intervals (PMI).
Tables
Data structure Type of test Test statistic p value N (mice) Figure 1B Normal distribution One-way ANOVA F(3,14) = 0.36 p = 0.786 18 Figure 1C Contingency table Chi-square test of independence X2 = 0.06, df = 6 p = 1 18 Figure 1-4 Contingency table Chi-square test of independence X2 = 4.26, df = 24 p = 1 18 Table 1 Normal distribution Two-sample t test t = 0.893, df = 16 p = 0.385 18 The Shapiro–Wilk test was applied to each group to assess normality. A one-way analysis of variance (ANOVA) was conducted to compare groups with normally distributed data. A comparison of RINe values between fresh 0 and 3 h postmortem intervals (PMI) was performed using a two-sample t test. Differences in cell type proportions across groups were analyzed using chi-square tests.
Figure 1-1
Sample genotype and sex verification. Log normalized gene expression of A) mouse Mapt, B) human MAPT, mouse C) Xist, and D) Uty. Download Figure 1-1, TIF file.
Figure 1-2
Single nucleus quality metrics do not differ among mice. A) Violin plots illustrating primary quality control (QC) metrics, including the number of counts per nucleus (nCount), detected features per nucleus (nFeature), cell complexity (the log10 ratio of nFeature to nCount), and the percentage of mitochondrial gene expression (percent mt) for PS19 0-hour (N = 4), WT 0-hour (N = 4), PS19 3-hour (N = 5), and WT 3-hour (N = 5) mice. Quality control metrics for each mouse for B) nCount, C) nFeature, D) cell complexity, and E) percent mt. Download Figure 1-2, TIF file.
Figure 1-3
Relative abundance of cell types among samples. A) the total nuclei count for each cell type is shown for both the 0-hour and 3-hour post-mortem interval (PMI) sample groups. B) the percentage of each cell type is presented across different genotypes (PS19 or WT) and PMI durations (0-hour or 3-hour). C) The proportion of each cell type within individual samples is illustrated, providing a detailed view of the distribution across all samples. Download Figure 1-3, TIF file.
Figure 1-4
Bubble plot showing brain cell type markers. The Y-axis represents different cell types, while the X-axis displays the corresponding biomarker genes. The size of each bubble indicates the percentage of nuclei within a given cell type that expresses that gene and the color represents the average log2 expression level. Download Figure 1-4, TIF file.
Figure 1-5
Principal component analysis (PCA) of aggregated expression values at the sample and cell type level. Clear delineation of the major cell types. Download Figure 1-5, TIF file.
Table 1-1
Metadata. Sample ID, genotype PS19 tau or wildtype (WT), sex, post-mortem interval (PMI) in hours, and RNA integrity number (RINe). Download Table 1-1, DOCX file.
Figure 3-1
The transcriptomic effect of PMI 3-hour versus 0-hour PMI for endothelial, mural, and microglia cell types. A) Volcano plot of differentially expressed genes (DEGs) in endothelial cell type, comparing 3-hour PMI (N = 10) versus fresh 0-hour (N = 8) samples (total N = 18). Downregulated genes (log2 fold change < 0, q < 0.1) are shown in green, upregulated genes (log2 fold change > 0, q < 0.1) in purple, and non-significant genes (q ≥ 0.1) in gray. Repeated for B) mural and C) microglia. Download Figure 3-1, TIF file.
Figure 4-1
Gene ontology (GO) enrichment analysis of upregulated and downregulated genes in PS19 versus WT at 0-hour and 3-hour post-mortem intervals (PMI). A) In the 0-hour PMI group, upregulated genes in PS19 neurons compared to WT are enriched in pathways such as nonsense-mediated decay, protein homooligomerization, steroid metabolism, neuronal systems, and endocytosis. The x-axis represents the GO term, and the y-axis, with the bar color indicating the −log10 p-value. B) Downregulated genes are enriched in pathways related to the RAF/MAP kinase cascade, head development, and cell cycle regulation. C) At 3-hour PMI, the overall signal of differentially expressed genes (DEGs) in PS19 versus WT was reduced, and no pathways were identified as upregulated due to an insufficient number of DEGs (q-value < 0.1). D) Downregulated genes at 3-hour PMI were enriched in supramolecular fiber organization. Download Figure 4-1, TIF file.
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