Directly Reprogrammed Human Neurons Retain Aging-Associated Transcriptomic Signatures and Reveal Age-Related Nucleocytoplasmic Defects

Abstract

Aging is a major risk factor for many human diseases, and in vitro generation of human neurons is an attractive approach for modeling aging-related brain disorders. However, modeling aging in differentiated human neurons has proved challenging. We generated neurons from human donors across a broad range of ages, either by iPSC-based reprogramming and differentiation or by direct conversion into induced neurons (iNs). While iPSCs and derived neurons did not retain aging-associated gene signatures, iNs displayed age-specific transcriptional profiles and revealed age-associated decreases in the nuclear transport receptor RanBP17. We detected an age-dependent loss of nucleocytoplasmic compartmentalization (NCC) in donor fibroblasts and corresponding iNs and found that reduced RanBP17 impaired NCC in young cells, while iPSC rejuvenation restored NCC in aged cells. These results show that iNs retain important aging-related signatures, thus allowing modeling of the aging process in vitro, and they identify impaired NCC as an important factor in human aging.

Figure 1. Age-Dependent Transcriptome Signatures in Fibroblasts and Derived iPSCs

(A) iPSC reprogramming of young and old donor fibroblasts using the four Yamanaka-factors (4YF). (B) Phase contrast images and immunocytochemical characterization of human iPSCs stained with Nanog. The scale bars represent 20 μm. (C) Representative image for karyotype analysis of iPSC clones with G-banding (all included lines tested normal). (D) Differentially expressed genes between fibroblasts and iPSCs. The top 100 up and downregulated genes are shown (extended data in Table S2). (E) Heatmap showing hierarchical clustering of transcriptome similarity of fibroblasts and derived iPSCs. The black bars indicate multiple iPSC clones from the same donor that cluster together, and the * indicates lines from the same donor that do not cluster together. (F and G) MA plot shows differential expression between young (<40 year) and old (>40 year) fibroblasts and iPSCs (one clone per donor used for analysis). The colored dots indicate significant aging genes (padj < 0.05) (extended data in Table S3).

Figure 2. Direct Conversion of Young and Old Human Fibroblasts into Functional iNs Is Efficient Regardless of Donor Age

(A) Direct iN conversion of young and old donor fibroblasts using Ngn2-2A-Ascl1 (N2A) and small molecular enhancers (SM). (B) Schematic of lentiviral system for inducible overexpression of N2A and SM for iN conversion. (C) Phase contrast images of progressively converting fibroblasts, and immunofluorescence images of 6-week converted fibroblasts stained with βIII-tubulin, hTau, NeuN, Map2ab, and vGlut1. (D) iNs co-cultured on astrocytes labeled with LV-hSyn∷RFP and stained for synapsin-I following 8 weeks of conversion. (E) Electrophysiological characterization of iNs shows multiple evoked and spontaneous action potentials. (F) Immunocytochemical characterization of iN from young and old donors after 3 weeks of conversion. All of the scale bars represent 20 μM. (G) Quantification of neuronal yields per DAPI from 18 donors following 3 weeks of conversion. The bar graphs show mean + SEM. (H) Electrophysiological characterization of young- and old-derived iNs shows Na⁺/K⁺ channel-mediated inward/outward currents in response to depolarizing voltage steps (upper) and multiple evoked action potentials (lower) without apparent differences between young and old (n = 13 donors). (I) Quantification of neuronal subtype markers based on immunocytochemical analysis (extended data in Figure S2). Also see related Figure S1.

Figure 3. FACS-Based Purification and Transcriptome Analysis of iNs

(A) Immunocytochemical staining of iNs with PSA-NCAM shows punctate staining on the surface of neurons. (B) Density plot of PSA-NCAM-stained iN cultures during FACSorting. (C) Quantification of βIII-tubulin and hTau-positive neurons over human nuclei (hNuc) 1 week after FACS purification and replating. The bar graphs showmean + SEM. (D) Immunocytochemical analysis of 3-week-old sorted neurons following a total of 8 weeks of conversion stained with βIII-tubulin, hTau, and human nuclei (hNuc). The scale bars represent 20 μm. (E) Heatmap of time course RNA-seq expression analysis of progressively converting iN cells and FACS-purified iNs (extended data in Table S4). (F) GO term analysis of the 200 most strongly (fold change) upregulated genes following 18 days of iN conversion (extended data in Table S5). (G)Gene expressionanalysis before andafterPSA-NCAMFACSpurification. The bars show normalized counts of day 18bulk (white) and day 18sorted (black) samples. Also see related Figure S2.

Figure 4. Age-Dependent Gene Expression in Human Fibroblasts and iNs

(A) Heatmap showing hierarchical clustering of transcriptome similarity of fibroblasts and derived iNs. The black bars indicate multiple independent conversions from the same donor. (B and C) Heatmaps of significantly differentially expressed genes between young (<40 year) and old (>40 year) FACS-purified fibroblasts (red; 78 genes) and iNs (blue; 202 genes) with an FDR-adjusted p value < 0.05 (extended data in Table S3). (D) Schematic Venn diagram showing the overlap of fibroblast and iN aging genes. (E and F) GO term and KEGG pathway analysis of the 78 fibroblast and 202 iN aging genes (extended data in Table S6). Also see related Figure S3.

Figure 5. Expression of RanBP17 in the Aging Human Prefrontal Cortex

(A) Schematic Venn diagram showing the overlap of postmortem cortex, fibroblast, and iN aging genes. (B) Correlation of RanBP17 expression with age in fibroblasts (red), purified iNs (blue), and RNA extracted from the human prefrontal cortex (green, n = 14 donors; Table S1). The data points indicate single donors, and the lines depict linear regression function (expression values: VST normalized counts). (C) Immunocytochemical analysis of subcellular RanBP17 localization in fibroblasts (upper) and iNs (lower) co-stained with hTau and Lamin B1. The scale bars represent 10 μm. (D) Formalin-fixed human cortices were embedded in paraffin and sectioned for immunohistochemistry to identify cortical neurons and layers. The βIII-tubulin-positive cortical neurons were brightly stained in cortices from young adult and old brains (look-up-table, LUT, colored; numbers indicate cortical layer structure). (E) Immunohistochemical analysis of young adult and old brains for βIII-tubulin and age-dependent decrease in RanBP17 in neurons. The scale bar represents 20 μm. (F) Representative images of RanBP17-stained cortical neurons show loss of RanBP17 from the neuronal somata and nuclei in old brains. (G) Quantification of neuronal fluorescence intensity comparing young and old brains (n = 10 donors). The bar graph shows means and dots individual cells (significance values: ****p < 0.001). (H) Quantification of western blot analysis for RanB17 protein over actin. The graph shows a correlation of cortical RanBP17 over actin with age. The dots indicate values of single donors ± SEM, and the line depicts linear regression. Also see related Figures S4 and S5.

Figure 6. NCC in Young and Old Fibroblasts and Age-Equivalent iNs

(A) 2Gi2R: lentiviral vector for expression of the IRES-linked NCC reporters 2xGFP:NES and 2xRFP:NLS. The experimental rationale for NCC measurement is that increasing GFP_nuc/RFP_nuc ratios indicate NCC defects and RFP_nuc/RFP_cyt and GFP_nuc/GFP_cyt ratios divide into import- or export-related phenotypes, respectively. (B) ROI selection of confocal sections for the measurement of nuclear GFP and RFP in cultured fibroblasts. (C) Representative florescence images of 2Gi2R reporter fibroblasts from a young adult (29 year) and old (69 year) donor. The scale bars represent 20 μm. (D)GFP_nuc/RFP_nuc, ratios in aging fibroblasts (n = 16 donors; normalized to young group). The bar graphs show mean + SEM and the dots indicate single cells (significance values: *p < 0.05; **p < 0.01; ***p < 0.005; and ****p < 0.001). (E) Correlation of NCC values of individual donors with age (black dots). The red line depicts linear regression fit, and the shaded area is the 95% confidence interval. (F) Experimental design: 3-week-old iNs were transduced with NCC reporter virus and relocated on a layer of astrocytes for maturation. (G) NCC was assessed by measuring neuronal GFP_nuc/RFP_nuc ratios in confocal sections. (H) GFP_nuc/RFP_nuc ratios in aging iNs (n = 14 donors). The data is normalized to the young group. The bar graphs show mean, and the dots indicate single cells (significance values: *p < 0.05; **p < 0.01; ***p < 0.005; and ****p < 0.001). (I) Correlation of NCC values of individual donors with age (black dots). The blue line depicts linear regression fit, and the shaded area is the 95% confidence interval. Also see related Figure S6.

Figure 7. RanBP17 Decrease Causes Loss of NCC and iPSC Reprogramming Restores NCC in Old Donor-Derived Cells

(A) Lentiviral vector for RanBP17 knockdown (shRNAs: iR#1 and iR#2). (B) Western immunoblotting for RanBP17 in cells expressing iR#1, iR#2, or scrambled control shRNA. (C) qPCR analysis for iR#1 and iR#2 compared to scrambled control. (D) GFP_nuc/RFP_nuc ratios in young (1 year, left) and old (71 year, right) RanBP17 knockdown cells. The bar graphs show mean, and the dots indicate single cells (significance values: *p < 0.05; **p < 0.01; ***p < 0.005, and ****p < 0.001). (E) Fibroblast aging genes in response to RanBP17 knockdown. The graph shows expression changes (old versus young) of the 78 fibroblast aging genes in response to aging (black bars) and in response to RanBP17 knockdown in young fibroblasts (1 year). The RanBP17 knockdown caused 68% of the aging genes to change in the same direction as in aging. (F) RanBP17 mRNA levels in fibroblasts (red) and corresponding iPSCs (green; VST normalized counts). (G) Monolayer PluriPro culture of iPSCs for the measurement of NCC. (H) GFP_nuc/RFP_nuc ratios in iPSCs reprogrammed from young and old donors (n = 12 donors). The bars represent means, and the dots represent individual cells. (I) Correlation of NCC with donor age in iPSCs. The black dots represent individual donor iPSCs. The green line represents the linear regression fit, and the shaded area is the 95% confidence interval. Also see related Figure S7.