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Transcriptional and epigenetic dysregulation impairs generation of proliferative neural stem and progenitor cells during brain aging

Nature Aging volume 4, pages 62–79 (2024)Cite this article

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Abstract

The decline in stem cell function during aging may affect the regenerative capacity of mammalian organisms; however, the gene regulatory mechanism underlying this decline remains unclear. Here we show that the aging of neural stem and progenitor cells (NSPCs) in the male mouse brain is characterized by a decrease in the generation efficacy of proliferative NSPCs rather than the changes in lineage specificity of NSPCs. We reveal that the downregulation of age-dependent genes in NSPCs drives cell aging by decreasing the population of actively proliferating NSPCs while increasing the expression of quiescence markers. We found that epigenetic deregulation of the MLL complex at promoters leads to transcriptional inactivation of age-dependent genes, highlighting the importance of the dynamic interaction between histone modifiers and gene regulatory elements in regulating transcriptional program of aging cells. Our study sheds light on the key intrinsic mechanisms driving stem cell aging through epigenetic regulators and identifies potential rejuvenation targets that could restore the function of aging stem cells.

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Fig. 1: Generation efficacy of proliferative NSPCs decreases in the aging brain.
Fig. 2: Downregulation of age-dependent genes drives NSPC aging.
Fig. 3: Age-dependent genes are potential rejuvenation targets for aging NSPCs.
Fig. 4: Changes in histone modifications at gene regulatory elements are related to transcriptional dysregulation of NSPC aging.
Fig. 5: Identification of the MLL complex as a key epigenetic regulator in the functional decline of aging NSPCs.
Fig. 6: Loss of function of the MLL complex results in transcriptional and epigenetic inactivation of age-dependent genes.

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Data availability

All the sequencing data have been submitted to the Gene Expression Omnibus and are publicly available as of the date of publication. The accession number for the sequencing data is GSE212725. The UniProtKB Mus musculus database was used for mass spectrometry data analysis (database link: https://www.uniprot.org/taxonomy/10090). All other data supporting the findings of this paper will be available from the corresponding author upon reasonable request. Source data are provided with this paper

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Acknowledgements

We thank D. Qin, Q. Shan and Z. Cai (Shantou University Medical College) for their technical support of stereotactic injection of mice. Thanks to B. Wang for technical support of animal studies. Thanks to X. Hu for technical support of NSPC isolation from the SVZ. Thanks to T. Su (University of California, Los Angeles) for bioinformatic support. Thanks to C. Peng and Y. Yin of the Mass Spectrometry System at the National Facility for Protein Science in Shanghai, Shanghai Advanced Research Institute, Chinese Academy of Sciences, for data collection and analysis. This work was supported by the National Natural Science Foundation of China (grant no. 81671396, to C.H.), the Natural Science Foundation of Guangdong Province (grant no. 2017A030313780, to C.H.) and a National Institutes of Health R01 grant (grant no. GM074701, to M.C.).

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  1. These authors contributed equally: Meiyang Li, Hongzhi Guo.

Authors and Affiliations

  1. Center for Neurobiology, Shantou University Medical College, Shantou, China

    Meiyang Li, Hongzhi Guo & Chengyang Huang

  2. Department of Biological Chemistry, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA, USA

    Michael Carey & Chengyang Huang

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Contributions

M.L. did most experiments and statistical analyses. H.G. performed the animal experiments. C.H. analyzed the bioinformatic data. C.H. and M.C. supervised the study.

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Correspondence to Michael Carey or Chengyang Huang.

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Extended data

Extended Data Fig. 1 Aged NSPCs exhibit reduced potential for differentiating into neurons compared to young NSPCs.

a, Confocal microscopy images showing immunofluorescence co-labeling of DCX and BrdU in the coronal SVZ sections from young and aged mice. Scale bar, 40 μm. The quantification of DCX and BrdU double-positive cells was performed in both the lateral and dorsolateral SVZ for each section. A total of four sections per mouse, spaced at 120 μm intervals along the rostrocaudal axis of the SVZ, were used for cell counting. Seven mice per young group. Six mice per aged group. The average number of DCX and BrdU double-positive cells per group was plotted in the chart on the right panel. P = 0.0009. **** P < 0.001. b, Immunofluorescence labeling of SOX2 in NSPCs isolated from the young and aged SVZ, respectively. Primary NSPC-derived neurospheres were dissociated into single cells for staining. Nuclei were stained with DAPI. Scale bar, 200 μm. Quantification data were plotted, showing the percentage of SOX2-positive cells among DAPI-positive cells counted from 10 microscopic fields per age group. ns: not significant.c, Immunofluorescence labeling of NESTIN in NSPCs isolated from the young and aged SVZ, respectively. The staining and quantification methods used are identical to those described in (b). d, The size of primary NSPC-derived neurospheres in young and aged groups. Neurospheres were imaged after 6 days of culture, and their diameters were measured through ImageJ software. n = 80. P = 3.05E-15. **** P < 0.001. e, Luminescent cell viability analysis of young and aged NSPCs cultured from Day 1 to Day 4. n = 3 independent experiments. Day1: P = 0.2436, Day2: P = 0.0012, Day3: P = 0.0013, Day4: P = 0.0003. *** P < 0.005. **** P < 0.001. ns: not significant. f, Cell death analysis by propidium iodide (PI) staining. Scale bar, 200 μm. Quantification data were plotted, showing the percentage of PI-positive cells among Hoechst-positive cells counted from 10 microscopic fields per age group. g, Neural differentiation starting from NSPC-derived neurosphere as neurite outgrows. After 2 days of differentiation, cells were fixed and subjected to immunofluorescence labeling of neuron marker TUJ1. The number of TUJ1+ differentiated cells with neurites that migrated away from the neurosphere was counted. Scale bar, 200 μm. Data were plotted with the percentage of TUJ1 versus DAPI-positive cells with neurites in each neurosphere differentiation. n = 10. P = 6.71E-05. h, Immunofluorescence labeling of neuron marker MAP2 four days post-differentiation. The number of MAP2+ differentiated cells that migrated away from the neurosphere was counted. Scale bar, 200 μm. n = 8 in young group. n = 13 in aged group. P = 4.83E-06. i, Immunofluorescence labeling of astrocyte marker GFAP four days post-differentiation. The number of GFAP+ differentiated cells that migrated away from the neurosphere was counted. Scale bar, 200 μm. Data were plotted with the percentage of GFAP-positive cells with astrocytic extensions versus DAPI-positive cells in each neurosphere differentiation. n = 13 per group. Data analysis for g-i: P = 3.99E-07. *** P < 0.005. **** P < 0.001. Error bars are mean ± SD in all graphs. Two-sided Student’s t-test for statistical analysis in all graphs.

Source data

Extended Data Fig. 2 Identification of NSPC activation states and lineage specificity through single-cell transcriptome.

a, Heatmap of top 10 differentially expressed marker genes in 4 clusters of NSPCs. Analysis was performed using merged scRNA-seq datasets of young and aged NSPCs. b, Ridgeline plots of merged scRNA-seq datasets of young and aged NSPCs, showing expression changes of activation markers from early type B1a cells to late type C cells. c, Violin plots showing the similar expression levels of NSPC lineage markers among 4 clusters between young and aged NSPCs. d, UMAP plots of NSPC lineage markers in young and aged NSPCs. e, Single-cell pseudotime analysis using Monocle3 shows dynamic transition of NSPC activation states along the trajectory. NSPCs with larger pseudotime values exhibit higher activity in proliferation. f, Gene ontology analysis of the DE markers of early type B1a cells. The gene number of each GO category is showed on the right side of the bar chart.

Source data

Extended Data Fig. 3 Transcriptional reversal of age-dependent genes is associated with the functional improvement of NSPCs in mice subjected to calorie restriction.

a, Percentage of BrdU and SOX2 double-positive versus SOX2-positive cells in the brain SVZs of young and aged mice subjected to AL and CR treatments. Seven mice per young group; eight mice per aged group. Twelve sections per mouse. Error bars are mean ± SD. Unpaired two-sided Student’s t-test. Young: P = 0.0146, Aged: P = 0.3789. * P < 0.05. ns: not significant. b, Quantification of DCX and BrdU double-positive cells in both the lateral and dorsolateral SVZ of young and aged mice subjected to AL and CR treatments. A total of four sections per mouse, spaced at 120 μm intervals along the rostrocaudal axis of the SVZ, were used for cell counting. Seven mice per young group. Six mice per aged group. Error bars are mean ± SD. Unpaired two-sided Student’s t-test. Young: P = 0.0024, Aged: P = 0.28. *** P < 0.005. ns: not significant. c, Representative images of neurospheres formed by primary NSPCs isolated from the SVZ of mice subjected to ad libitum (AL) feeding or calorie restriction (CR) within each age group. Scale bar, 1000 μm. The SVZ tissues were pooled from five mice per group. d, Number of neurospheres was quantified across four groups of mice as in (a). n = 3 independent cultures. Six microscopic fields were imaged from each culture replicate for cell counting. Error bars are mean ± SD. Unpaired two-sided Student’s t-test. Young_AL vs. Young_CR: P = 0.0032, Young_AL vs. Aged_AL: P = 0.0009, Aged_AL vs. Aged_CR: P = 0.5534. *** P < 0.005. **** P < 0.001. ns: not significant. e, f, Heatmap showing the fold change in transcriptional levels of age-dependent genes (Aged vs. Young, FC > 1.5) and their transcriptional reversal in young (c) or aged (d) mice subjected to calorie restriction (CR vs. AL, FC > 1.5).

Source data

Extended Data Fig. 4 Downregulation of age-dependent gene upregulates NSC quiescence markers.

a, Heatmap showing scRNA-seq differential expression analysis of the age-dependent genes identified in bulk RNA-seq in each NSPC cluster. Data are the average log2 fold change of expression. b, Heatmap showing bulk RNA-seq differential expression analysis of DE genes detected in early type B1a cells as in Fig. 1j. Fold change of RNA level (Aged_FPKM vs. Young_FPKM) and p-value were plotted. c, d, Quantitative real-time PCR analysis of relative expression (siRNA group vs. siControl group) of Aldoc and Atp1a2 in young NSPCs 48 hours after siRNA transfection. n = 3 independent assays. Error bars are mean ± SD. Unpaired two-sided Student’s t-test.

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Extended Data Fig. 5 The rejuvenating effects of age-dependent genes on aged NSPCs.

a, Quantification of SOX2-positive cells in the SVZ. The average percentage of SOX2+ /DAPI+ cells was plotted based on data from three aged wild-type mice. A total of eight sections per mouse, spaced at 120 μm intervals along the rostrocaudal axis of the SVZ, were used for cell counting. Cells were quantified within the lateral and dorsolateral SVZ in each section. b–d, Quantification of BrdU+SOX2+ NSPCs in the left (Igf2bp2, Igf2bp3, and Barhl2 overexpression) and right (wild-type) sides of the SVZ. The adenovirus encoding these genes were injected in the left side of SVZ of aged mice, respectively. BrdU labeling was performed two weeks after the gene transduction. BrdU+SOX2+ NSPCs were quantified at both sides of the lateral and dorsolateral SVZ in each section. Seven sections per mouse. Three mice per group. Unpaired two-sided Student’s t-test. (b): P = 0.0396, (c): P = 5.05E-09, (d): P = 1.39E-08. * P < 0.05. **** P < 0.001. e, f, Overexpression of Igf2bp2 and Igf2bp3 increases the number of DCX-positive cells in the SVZ of aged mice. Control mice were injected with control adenovirus without expressing these genes. Confocal microscopy images showing immunofluorescence co-labeling of DCX and DAPI in the SVZ. Scale bar, 20 μm. DCX-positive cells were quantified at the injection site within the lateral and dorsolateral SVZ in each section. A total of six sections per mouse, spaced at 120 μm intervals along the rostrocaudal axis of the SVZ, were used for cell counting. Three mice per group. The average number of DCX-positive cells per group was plotted blow the images. Unpaired two-sided Student’s t-test. (e): P = 0.0355, (f): P = 0.0216. * P < 0.05. g–i, Quantitative real-time PCR analysis of relative expression of Igf2bp2, Igf2bp3 and Barhl2 40 hours after the adenovirus-mediated transduction of the respective genes in primary aged NSPCs. The control group was transduced with control adenovirus without expressing these genes. n = 3 independent assays. Unpaired two-sided Student’s t-test. j, Cell death analysis by propidium iodide (PI) staining in primary aged NSPCs transduced with the genes of Igf2bp2, Igf2bp3 and Barhl2. NSPC-formed neurospheres were dissociated into single cells for staining 48 hours following adenovirus-mediated transduction of the respective genes. Scale bar, 200 μm. k, Quantification data of PI staining shows the percentage of PI-positive cells among Hoechst-positive cells, which were counted from 10 microscopic fields per group. Error bars are mean ± SD for b-k.

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Extended Data Fig. 6 Changes in average profiles of histone marks and ATAC-seq signals at promoters and enhancers in aging NSPCs.

a, Average profile of H3K4me3 and H3K27me3 at the promoters of all genes spanning TSSs in young and aged NSPCs (Two-sided Wilcoxon rank sum test, H3K4me3 data p-value = 0.2209, H3K27me3 data p-value = 6.69E-15). b, Average profile of H3K27ac signals spanning the center of enhancers of all genes in young and aged NSPCs (Two-sided Wilcoxon rank sum test, H3K27ac data p-value = 0.2684). c, Pearson correlation analysis of fold changes of RNA levels and promoter H3K4me3 signals of age-dependent genes. 95% confidence interval. d, Pearson correlation analysis of fold changes of RNA levels and enhancer H3K27ac signals of age-dependent genes. 95% confidence interval. e, f, Average profiles of ATAC- seq at promoters and enhancers of all genes show no genome-wide changes of chromatin accessibility across the regulatory elements during NSPC aging (Two-sided Wilcoxon rank sum test, promoter profile p-value = 0.9261, enhancer profile p-value = 0.6969).

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Extended Data Fig. 7 Histone modifications are associated with chromatin accessibility at the promoters of age-dependent genes in aging NSPCs.

a, Heatmap shows fold change (Aged vs. Young) of RNA levels of age-dependent genes and the average signals of ATAC-seq at their regulatory elements. b, Venn diagrams show the overlap of age-dependent genes that have decreased levels of chromatin accessibility and H3K4me3 at the promoters during NSPC aging. Two-sided Hypergeometric test for significant overlap analysis, * p-value = 5.602e-26. c, Venn diagrams show the overlap of age- dependent genes that have decreased levels of chromatin accessibility and H3K27ac signals at the enhancers during NSPC aging. Two-sided Hypergeometric test, * p-value = 3.618e-16. d, Browser tracks of ATAC-seq and ChIP-seq show a similar level of signals at the promoters of NSPC lineage markers and housekeeping genes between the young and aged groups. e, Browser tracks of 3 age-dependent down-regulated genes (Igf2bp3, Twist1 and Otx2) and 2 up-regulated genes (Gpr37l1 and Ifi27) that have changes of ATAC-seq signals and histone modifications at the promoters during NSPC aging.

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Extended Data Fig. 8 Age-related changes in histone modifications and transcription in aging EGFR+ NSPCs.

a, FACS plots of the isolation of EGFR-positive and 7-AAD negative NSPCs from the SVZ of young and aged mice. b, Average profile of H3K4me3 at the promoters of all genes spanning TSSs in young and aged EGFR+ NSPCs (Two-sided Wilcoxon rank sum test, H3K4me3 data p-value = 0.5428). c, Browser tracks of CUT&Tag signals of H3K4me3 at the promoters of NSPC lineage markers in young and aged EGFR+ NSPCs. d, Heatmap showing the fold change of average CUT&Tag signals of H3K4me3 at each promoter of age-dependent genes in aged NSPC-derived neurospheres or EGFR+ NSPCs compared to their young counterparts (Aged vs. Young, FC > 1.5). e, Browser tracks of CUT&Tag signals at the promoters of Barhl2, Igf2bp2, and Igf2bp3. f, Real-time qPCR analysis of relative expression of genes in aged versus young EGFR+ NSPCs. Error bars are mean ± SD. n = 3 independent assays. Two-sided Student’s t-tests. Otx2: P = 0.0021, Rimbp2: P = 0.0003, Twist1: P = 0.0099, Igf2bp3: P = 7.77E-06, Barhl2: P = 0.0001, Fam155a: P = 0.0382, Epb41l13: P = 0.0109, Fn3k: P = 0.0174, Igf2bp2: P = 7.57E-05, Angpt2: P = 0.0035. * P < 0.05. ** P < 0.01. *** P < 0.005. **** P < 0.001.

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Extended Data Fig. 9 Cell death analysis by propidium iodide (PI) staining in NSPCs following knockdown of MLL complex subunits.

a, Representative immunofluorescence images showing PI staining in siRNA-transfected young NSPCs. NSPC-derived neurospheres were dissociated into single cells for PI staining 48 hours post-transfection of siRNAs. Hoechst (blue) staining visualizes the nuclei. Scale bar, 200 μm. b, Quantification of PI staining in siRNA-transfected young NSPCs, displaying the percentage of PI-positive cells among Hoechst-positive cells. The quantification was performed by counting from 10 microscopic fields per group. Error bars are mean ± SD.

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Extended Data Fig. 10 Interaction between MLL complex and Cbx3 in aging NSPCs.

a, Mass spectrometry peptide counts (number of unique peptides) of MLL complex components identified in KMT2D immunoprecipitation product from young NSPC nuclear extract. b, Mass spectrometry peptide counts (number of unique peptides) of proteins identified in CBX3 immunoprecipitation product from young NSPC nuclear extract. c, Western blot analysis of endogenous MLL complex components in CBX3 and IgG immunoprecipitation products from the young and aged NSPC nuclear extracts. Bar charts show the relative levels of the indicated proteins in aged versus young IP groups analyzed by Image J software. Error bars are mean ± SD. n = 2 independent experiments, Unpaired two-sided Student’s t-tests. KMT2D: P = 0.0222, RBBP5: P = 0.0015, WDR5: P = 0.6499, CBX3: P = 0.4363. * P < 0.05. *** P < 0.005. ns: not significant.

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Li, M., Guo, H., Carey, M. et al. Transcriptional and epigenetic dysregulation impairs generation of proliferative neural stem and progenitor cells during brain aging. Nat Aging 4, 62–79 (2024). https://doi.org/10.1038/s43587-023-00549-0

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