Extended Data Fig. 4: Continuous decrease in neurogenic activity is a hallmark of neurogenic aging.
a, Projection of module score of Waterfall-defining TFs in pseudotemporal trajectory of qNSCs. Shading indicates 95% confidence interval. b, Alluvial plot between (left) real-time and (right) pseudotime gene expression trajectories of qNSC during aging shows age-dependent upregulation gene modules (in red) largely overlap with qNSC-enriched pseudotime gene modules while age-dependent downregulation gene modules (in blue) largely overlap with fate-committed population-enriched pseudotime gene modules. The line plots of the real-time gene expression trajectories depict the standardized gene expression signals, with each pink line representing the signal of the same gene in different cell populations and the black lines indicating the mean values for each gene cluster. c, Schematic overview of the Neurogenic Aging Signature calculation. d, Venn diagram indicating the overlap between the real-time and pseudotime trajectories. Upper: Between Real-age (real-time) upregulation and functional-age (pseudotime) downregulation; Lower: Between Real-age (real-time) downregulation and functional-age (pseudotime) upregulation. e, Module scores of Neurogenic Aging Signature of qNSCs. Upper: Neurogenic Aging Signature-UP score of qNSCs (two-tailed unpaired t-test with Welch’s correction; t = 6.217, ****P < 0.0001 between young and middle-age, t = 3.739, ***P = 0.0003 between middle-age and old, t = 9.396, ****P < 0.0001 between young and old; young, n = 141 cells, middle-age, n = 84 cells, old, n = 69 cells). Lower: Neurogenic Aging Signature-DOWN score of qNSCs (two-tailed unpaired t-test with Welch’s correction; t = 6.843, ****P < 0.0001 between young and middle-age, t = 4.930, ****P < 0.0001 between middle-age and old, t = 13.55, ****P < 0.0001 between young and old; young, n = 141 cells, middle-age, n = 84 cells, old, n = 69 cells). Box plots depict the median and interquartile range, with whiskers indicating minimum and maximum values. f, GO terms enriched in (upper) Neurogenic Aging Signature-UP and (lower) Neurogenic Aging Signature-DOWN. All GO terms shown by an adjusted P < 0.05 with Benjamini–Hochberg correction. g, Pie charts of the percentage of TFs in Neurogenic Aging Signature (upper) -UP and (lower) -DOWN. h, Spatial feature plots of (left) Neurogenic Aging Signature-UP and (right) Neurogenic Aging Signature-DOWN showing regional-specific expression patterns in hippocampus. Color gradient indicates the expression level of individual module score. i, Venn diagram indicating the overlap between (upper) the Astrocyte-enriched genes and Neurogenic Aging Signature-UP and (lower) the qNSC-enriched genes and Neurogenic Aging Signature-DOWN. j, Expression of Astrocyte- and qNSC-enriched genes in qNSCs among different ages. Upper: Module scores of the Astrocyte-enriched genes in qNSC (two-tailed unpaired t-test with Welch’s correction; t = 5.241, ****P < 0.0001 between young and middle-age, t = 2.462, *P = 0.0151 between middle-age and old, t = 7.172, ****P < 0.0001 between young and old; young, n = 141 cells, middle-age, n = 84 cells, old, n = 69 cells) and expression of selected Astrocyte-enriched genes, Grm3, Slc15a2 and Bcan. Lower: Module scores of the qNSC-enriched genes in qNSC (two-tailed unpaired t-test with Welch’s correction; t = 5.035, ****P < 0.0001 between young and middle-age, t = 3.037, **P = 0.0028 between middle-age and old, t = 8.787, ****P < 0.0001 between young and old; young, n = 141 cells, middle-age, n = 84 cells, old, n = 69 cells). Box plots depict the median and interquartile range, with whiskers indicating minimum and maximum values. k, UMAP plot of neurogenic lineages in Hochgerner 2018 dataset. l, Module scores of Neurogenic Aging Signature of RGL. Upper: Neurogenic Aging Signature-UP score of RGL (two-tailed unpaired t-test with Welch’s correction; t = 12.79, ****P < 0.0001 between perinatal and juvenile, t = 1.098, NS, P = 0.2745 between juvenile and adult, t = 9.396, ****P < 0.0001 between perinatal and adult; perinatal, n = 461 cells, juvenile, n = 57 cells, adult, n = 127 cells). Lower: Neurogenic Aging Signature-DOWN score of RGL (two-tailed unpaired t-test with Welch’s correction; t = 16.10, ****P < 0.0001 between perinatal and juvenile, t = 3.501, ***P = 0.0007 between juvenile and adult, t = 25.22, ****P < 0.0001 between perinatal and adult; perinatal, n = 461 cells, juvenile, n = 57 cells, adult, n = 127 cells). Box plots depict the median and interquartile range, with whiskers indicating minimum and maximum values. m, Immunofluorescent staining of radial NSCs (GFAP+SOX2+S100b− cells with radial morphology) and newly born neuronal progeny (DCX+ cells). n, Quantification of (upper) the number of radial NSC (young: 11151 ± 375 cells; middle-age: 3923 ± 200 cells; old: 1324 ± 154 cells; very old: 934 ± 100 cells; two-tailed unpaired t-test with Welch’s correction, ****P < 0.0001 between young and middle-age, ****P < 0.0001 between middle-age and old, NS, P = 0.0558 between old and very old), (middle) the number of neuroblast (young: 12131 ± 392 cells; middle-age: 1079 ± 32 cells; old: 173 ± 25 cells; two-tailed unpaired t-test with Welch’s correction, ****P < 0.0001 between young and middle-age, ****P < 0.0001 between middle-age and old, ****P < 0.0001 between old and very old) and (lower) the ratio of radial NSCs to neuroblasts from young (3 MO) to very old (24-25 MO) (young: 0.9 ± 0.0; middle-age: 3.7 ± 0.2; old: 8.3 ± 0.7; very old: 16.7 ± 1.3; two-tailed unpaired t-test with Welch’s correction, ****P < 0.0001 between young and middle-age, ****P < 0.0001 between middle-age and old, ****P < 0.0001 between old and very old). aNSPC, active neural stem and progenitor cells, GO, gene ontology, GRN, gene regulatory network, IPC, intermediate progenitor cell, MO, month-old, NB/IMN, neuroblast and immature neuron, NS, not significant, NSC, neural stem cell, SGZ, subgranular zone, TF, transcription factor. All data were presented as mean ± SEM. Scale bars, 20 μm.
