Extended Data Fig. 5: Validation of downregulation during ageing.

(a) Expression of genes downregulated in the primary analysis (elderly vs. adult) are also downregulated when data is broken down into three groups, donors 15–39 (red, N = 5), donors 40–69 (magenta, N = 6), and donors 70–104 (purple, N = 6). Some cell types exhibit continuous downregulation, showing significant decreases with each age group while others are significantly downregulated between the 15–39- and 40–69-year-old groups but expression does not change between the older adult and elderly groups (Two-sided Wilcoxon rank-sum test). (b) Volcano plots showing the results of expression changes during ageing, determined by linear regression. Regression slope is shown on the x axis and -log₁₀(p-value) on the y axis. Dotted lines indicate slope and p-value thresholds, slope < −0.001 or > 0.001 and p < 0.05, used to determine significance. In addition, genes had to be expressed in at least 25% of the elderly or adult cells to be considered. Blue dots indicate genes that were also identified as significantly downregulated by pairwise comparison and Two-sided Wilcoxon rank-sum test. Red dots indicate genes that were identified as significantly upregulated by pairwise comparison and Wilcoxon test. Open circles indicate genes that did not meet the pairwise comparison criteria for fold change and grey circles indicate genes that met the fold change criteria but did not have significant p-values in the pairwise comparison. (c) Box plots showing the expression, in log(CPM), of significantly downregulated genes in elderly excitatory neurons identified in this study in our donors (left) with the donors from Ling et al.²⁹ (right). (d) Box plots comparing expression of downregulated genes identified in this study in adults from this study (red, N = 9), all donors from Mathys et al.³⁰ (violet, N = 189), elderly donors from this study (lilac, N = 7), donors 70–79 from Mathys et al. (light purple, N = 34), and donors over 80 from Mathys et al. (dark purple, N = 155). Two-sided Wilcoxon rank-sum test comparing adults in this study to each of the Mathys groups are all significant. All box plots depict median, and first and third quartile. Whiskers show 1.5 × IQR beyond the first and third quartiles. (*, p > 0.05; **, p > 0.01; ***, p > 0.001).